Augmented Acid Alpha-Glucosidase  For The Treatment Of Pompe Disease

ABSTRACT

A method for treating Pompe disease including administration of recombinant human acid α-glucosidase having optimal glycosylation with mannose-6-phosphate residues in combination with an amount of miglustat effective to maximize tissue uptake of recombinant human acid α-glucosidase while minimizing inhibition of the enzymatic activity of the recombinant human acid α-glucosidase is provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) to U.S.Provisional Application No. 62/272,890 filed Dec. 30, 2015, U.S.Provisional Application No. 62/300,479 filed Feb. 26, 2016, U.S.Provisional Application No. 62/315,412 filed Mar. 30, 2016, U.S.Provisional Application No. 62/402,454 filed Sep. 30, 2016, U.S.Provisional Application No. 62/428,867 filed Dec. 1, 2016 and U.S.Provisional Application No. 62/431,791 filed Dec. 8, 2016, the entirecontents of which are incorporated herein by reference in theirentirety.

FIELD

The present invention provides a method for treating Pompe diseasecomprising administering to an individual a combination of an acidα-glucosidase and a pharmacological chaperone thereof. Morespecifically, the present invention provides a method for treating Pompedisease comprising administering to an individual a combination ofrecombinant human acid α-glucosidase and miglustat.

BACKGROUND

Pompe disease, also known as acid maltase deficiency or glycogen storagedisease type II, is one of several lysosomal storage disorders.Lysosomal storage disorders are a group of autosomal recessive geneticdiseases characterized by the accumulation of cellularglycosphingolipids, glycogen, or mucopolysaccharides withinintracellular compartments called lysosomes. Individuals with thesediseases carry mutant genes coding for enzymes which are defective incatalyzing the hydrolysis of one or more of these substances, which thenbuild up in the lysosomes. Other examples of lysosomal disorders includeGaucher disease, G_(M1)-gangliosidosis, fucosidosis,mucopolysaccharidoses, Hurler-Scheie disease, Niemann-Pick A and Bdiseases, and Fabry disease. Pompe disease is also classified as aneuromuscular disease or a metabolic myopathy.

Pompe disease is estimated to occur in about 1 in 40,000 births, and iscaused by a mutation in the GAA gene, which codes for the enzymelysosomal α-glucosidase (EC:3.2.1.20), also commonly known as acidα-glucosidase. Acid α-glucosidase is involved in the metabolism ofglycogen, a branched polysaccharide which is the major storage form ofglucose in animals, by catalyzing its hydrolysis into glucose within thelysosomes. Because individuals with Pompe disease produce mutant,defective acid α-glucosidase which is inactive or has reduced activity,glycogen breakdown occurs slowly or not at all, and glycogen accumulatesin the lysosomes of various tissues, particularly in striated muscles,leading to a broad spectrum of clinical manifestations, includingprogressive muscle weakness and respiratory insufficiency. Tissues suchas the heart and skeletal muscles are particularly affected.

Pompe disease can vary widely in the degree of enzyme deficiency,severity and age of onset, and over 500 different mutations in the GAAgene have been identified, many of which cause disease symptoms ofvarying severity. The disease has been classified into broad types:early onset or infantile and late onset. Earlier onset of disease andlower enzymatic activity are generally associated with a more severeclinical course. Infantile Pompe disease is the most severe, resultingfrom complete or near complete acid α-glucosidase deficiency, andpresents with symptoms that include severe lack of muscle tone,weakness, enlarged liver and heart, and cardiomyopathy. The tongue maybecome enlarged and protrude, and swallowing may become difficult. Mostaffected children die from respiratory or cardiac complications beforethe age of two. Late onset Pompe disease can present at any age olderthan 12 months and is characterized by a lack of cardiac involvement andbetter short-term prognosis. Symptoms are related to progressiveskeletal muscle dysfunction, and involve generalized muscle weakness andwasting of respiratory muscles in the trunk, proximal lower limbs, anddiaphragm. Some adult patients are devoid of major symptoms or motorlimitations. Prognosis generally depends on the extent of respiratorymuscle involvement. Most subjects with Pompe disease eventually progressto physical debilitation requiring the use of a wheelchair and assistedventilation, with premature death often occurring due to respiratoryfailure. Recent treatment options for Pompe disease include enzymereplacement therapy (ERT) with recombinant human acid α-glucosidase(rhGAA). Conventional rhGAA products are known under the namesalglucosidase alfa, Myozyme® or Lumizyme®; Genzyme, Inc. ERT is achronic treatment required throughout the lifetime of the patient, andinvolves administering the replacement enzyme by intravenous infusion.The replacement enzyme is then transported in the circulation and enterslysosomes within cells, where it acts to break down the accumulatedglycogen, compensating for the deficient activity of the endogenousdefective mutant enzyme, and thus relieving the disease symptoms. Insubjects with infantile onset Pompe disease, treatment withalglucosidase alfa has been shown to significantly improve survivalcompared to historical controls, and in late onset Pompe disease,alglucosidase alfa has been shown to have a statistically significant,if modest, effect on the 6-Minute Walk Test (6MWT) and forced vitalcapacity (FVC) compared to placebo.

However, the majority of subjects either remain stable or continue todeteriorate while undergoing treatment with alglucosidase alfa. Thereason for the apparent sub-optimal effect of ERT with alglucosidasealfa is unclear, but could be partly due to the progressive nature ofunderlying muscle pathology, or the poor tissue targeting of the currentERT. For example, the infused enzyme is not stable at neutral pH,including at the pH of plasma (about pH 7.4), and can be irreversiblyinactivated within the circulation. Furthermore, infused alglucosidasealfa shows insufficient uptake in key disease-relevant muscles, possiblydue to inadequate glycosylation with mannose-6-phosphate (M6P) residues.Such residues bind cation-independent mannose-6-phosphate receptors(CIMPR) at the cell surface, allowing the enzyme to enter the cell andthe lysosomes within. Therefore, high doses of the enzyme may berequired for effective treatment so that an adequate amount of activeenzyme can reach the lysosomes, making the therapy costly andtime-consuming.

In addition, development of anti-recombinant human acid α-glucosidaseneutralizing antibodies often develop in Pompe disease patients, due torepeated exposure to the treatment. Such immune responses can severelyreduce the tolerance of patients to the treatment. The US product labelfor alglucosidase alfa includes a black box warning with information onthe potential risk of hypersensitivity reaction. Life-threateninganaphylactic reactions, including anaphylactic shock, have been observedin subjects treated with alglucosidase alfa.

Next-generation ERT is being developed to address these shortcomings. Inone strategy, recombinant enzymes can be co-administered withpharmacological chaperones which can induce or stabilize a properconformation of the enzyme, to prevent or reduce degradation of theenzyme and/or its unfolding into an inactive form, either in vitro (forexample, in storage prior to administration) or in vivo. Such a strategyis described in International Patent Application Publications No. WO2004/069190, WO 2006/125141, WO 2013/166249 and WO 2014/014938.

The results of clinical trials of co-administration of alglucosidasealfa with miglustat to patients with Pompe disease have been described.In a clinical trial conducted in 13 subjects with Pompe disease (3 earlyonset (infantile) and 10 late onset) at 4 treatment centers in Italy, 20to 40 mg/kg alglucosidase alfa was administered alone and thenco-administered with 4 doses of 80 mg miglustat. The results of thestudy showed a mean 6.8-fold increase in acid α-glucosidase activityexposure (measured in terms of the pharmacokinetic parameter AUC (areaunder the concentration v. time curve)) for co-administration comparedto alglucosidase alfa alone (Parenti, G., G. Andria, et al. (2015).“Lysosomal Storage Diseases: From Pathophysiology to Therapy.” Annu.Rev. Med. 66(1): 471-486). In addition, a study conducted at theUniversity of Florida evaluated the pharmacokinetics (PK) of plasmamiglustat when co-administered with intravenous infusion ofalglucosidase alfa to subjects with Pompe disease (Doerfler, P. A., J.S. Kelley, et al. (2014). “Pharmacological chaperones prevent theprecipitation of rhGAA by anti-GAA antibodies during enzyme replacementtherapy.” Mol. Genet. Metab. 111(2): S38).

However, there remains a need for further improvements to enzymereplacement therapy for treatment of Pompe disease. For example, newrecombinant human acid α-glucosidase enzymes are desirable which canhave one or more advantages over presently used enzymes, including butnot limited to improved tissue uptake, improved enzymatic activity,improved stability or reduced immunogenicity.

SUMMARY

The present invention provides a method of treating Pompe disease in apatient in need thereof, the method including administering miglustat tothe patient in combination with a recombinant human acid α-glucosidase(rhGAA), wherein the recombinant human acid α-glucosidase is expressedin Chinese hamster ovary (CHO) cells and comprises an increased contentof N-glycan units bearing one or two mannose-6-phosphate residues whencompared to a content of N-glycan units bearing one or twomannose-6-phosphate residues of alglucosidase alfa. In at least oneembodiment, the recombinant human acid α-glucosidase is administeredintravenously at a dose of about 20 mg/kg and the miglustat isadministered orally at a dose of about 260 mg.

In another aspect, the present invention provides a combination ofmiglustat and a recombinant human acid α-glucosidase as defined hereinfor the treatment of Pompe disease in a patient in need thereof.

In another aspect, the present invention provides the use of acombination of miglustat and a recombinant human acid α-glucosidase asdefined herein in the preparation of an agent for the treatment of Pompedisease in a patient in need thereof

Another aspect of the present invention provides a kit for combinationtherapy of Pompe disease in a patient in need thereof, the kit includinga pharmaceutically acceptable dosage form comprising miglustat, apharmaceutically acceptable dosage form comprising a recombinant humanacid α-glucosidase as defined herein, and instructions for administeringthe pharmaceutically acceptable dosage form comprising miglustat and thepharmaceutically acceptable dosage form comprising the recombinant acidα-glucosidase to a patient in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features of the present invention will become apparent from thefollowing written description and the accompanying figures, in which:

FIG. 1 is a graph showing the percentage of unfolded ATB200 protein atvarious pH values and in the presence and absence of miglustat vs.temperature;

FIGS. 2A and 2B, respectively, show the results of CIMPR affinitychromatography of Lumizyme® and Myozyme®. The dashed lines refer to theM6P elution gradient. Elution with M6P displaces GAA molecules bound viaan M6P containing glycan to CIMPR. As shown in FIG. 2A, 78% of the GAAactivity in Lumizyme® eluted prior to addition of M6P. FIG. 2B showsthat 73% of the GAA Myozyme® activity eluted prior to addition of M6P.Only 22% or 27% of the rhGAA in Lumizyme® or Myozyme®, respectively, waseluted with M6P. These figures show that most of the rhGAA in these twoconventional rhGAA products lack glycans having M6P needed for cellularuptake and lysosomal targeting.

FIG. 3 shows a DNA construct for transforming CHO cells with DNAencoding rhGAA. CHO cells were transformed with a DNA construct encodingrhGAA.

FIGS. 4A and 4B, respectively show the results of CIMPR affinitychromatography of Myozyme® and ATB200 rhGAA. As apparent from FIG. 4B,about 70% of the rhGAA in ATB200 rhGAA contained M6P.

FIGS. 5A and 5B show the results of CIMPR affinity chromatography ofATB200 rhGAA with and without capture on an anion exchange (AEX) column.

FIG. 6 shows Polywax elution profiles of Lumizyme® and ATB200 rhGAAs.

FIG. 7 shows a summary of N-glycan structures of Lumizyme® compared tothree different preparations of ATB200 rhGAA, identified as BP-rhGAA,ATB200-1 and ATB200-2.

FIGS. 8A-8H show the results of a site-specific N-glycosylation analysisof ATB200 rhGAA.

FIG. 9A compares the CIMPR binding affinity of ATB200 rhGAA (left trace)with that of Lumizyme® (right trace).

FIG. 9B compares the Bis-M6P content of Lumizyme® and ATB200 rhGAA.

FIG. 10A compares ATB200 rhGAA activity (left trace) with Lumizyme®rhGAA activity (right trace) inside normal fibroblasts at various GAAconcentrations.

FIG. 10B compares ATB200 rhGAA activity (left trace) with Lumizyme®rhGAA activity (right trace) inside fibroblasts from a subject havingPompe Disease at various GAA concentrations.

FIG. 10C compares (K_(uptake)) of fibroblasts from normal subjects andsubjects with Pompe Disease.

FIG. 11 is a graph showing goodness of fit of a populationpharmacokinetic (PK) model for ATB200;

FIG. 12 is a graph showing dose-normalized plasma concentration-timeprofiles of miglustat and duvoglustat;

FIG. 13A is a graph showing goodness of fit of a population PK model forduvoglustat in plasma;

FIG. 13B is a graph showing goodness of fit of a population PK model forduvoglustat in muscle tissue;

FIG. 14 is a graph showing goodness of fit of a population PK model formiglustat;

FIG. 15 is a graph showing the predicted concentration-time profileresulting from infusion of a single 20 mg/kg intravenous (IV) dose ofATB200 in humans over a 4 h period;

FIG. 16A is a graph showing the amount of glycogen relative to dose ofrecombinant human acid α-glucosidase in mouse heart muscle after contactwith vehicle (negative control), with 20 mg/kg alglucosidase alfa(Lumizyme®), or with 5, 10 or 20 mg/kg ATB200;

FIG. 16B is a graph showing the amount of glycogen relative to dose ofrecombinant human acid α-glucosidase in mouse quadriceps muscle aftercontact with vehicle (negative control), with 20 mg/kg alglucosidasealfa (Lumizyme®), or with 5, 10 or 20 mg/kg ATB200;

FIG. 16C is a graph showing the amount of glycogen relative to dose ofrecombinant human acid α-glucosidase in mouse triceps muscle aftercontact with vehicle (negative control), with 20 mg/kg alglucosidasealfa (Lumizyme®), or with 5, 10 or 20 mg/kg ATB200;

FIG. 17 is a graph plotting the ratio of glycogen levels in mice treatedwith varying doses of miglustat in the presence of ATB200 to glycogenlevels in mice treated with ATB200 alone against the ratio of the AUCvalue of miglustat to the AUC value of ATB200;

FIG. 18 is a graph showing the predicted concentration-time profile ofmiglustat in plasma following repeated dosing of doses of 466 mg, 270 mgand 233 mg of miglustat;

FIG. 19 is a graph showing the predicted concentration-time profile ofmiglustat in tissue lysosomes following repeated dosing of doses of 466mg, 270 mg and 233 mg of miglustat;

FIG. 20 is a series of photomicrographs of heart, diaphragm and soleusmuscle from wild-type and Gaa-knockout mice treated with vehicle,alglucosidase alfa and ATB200 in the presence and absence of miglustat,showing levels of lysosome associated membrane protein (LAMP1);

FIG. 21 is a series of photomicrographs of heart and soleus muscle fromwild-type and Gaa-knockout mice treated with vehicle, alglucosidase alfaand ATB200 in the presence and absence of miglustat, showing glycogenlevels by staining with periodic acid-Schiff reagent (PAS);

FIG. 22 is a series of photomicrographs (1000×) of quadriceps musclefrom wild-type and Gaa-knockout mice treated with vehicle, alglucosidasealfa and ATB200 in the presence and absence of miglustat, stained withmethylene blue to show vacuoles (indicated by arrows);

FIG. 23 is a series of photomicrographs (400×) of quadriceps muscle fromwild-type and Gaa-knockout mice treated with vehicle, alglucosidase alfaand ATB200 in the presence and absence of miglustat, showing levels ofthe autophagy markers microtubule-associated protein 1A/1B-light chain 3phosphatidylethanolamine conjugate (LC3A II) and p62, theinsulin-dependent glucose transporter GLUT4 and the insulin-independentglucose transporter GLUT1;

FIGS. 24A-24D are graphs showing the concentration-time profiles of GAAactivity in plasma in human subjects after dosing of 5, 10 or 20 mg/kgATB200, or 20 mg/kg ATB200 and 130 or 260 mg miglustat;

FIGS. 25A-25D are graphs showing the concentration-time profiles of GAAtotal protein in plasma in human subjects after dosing of 5, 10 or 20mg/kg ATB200, 20 mg/kg ATB200 and 130 mg miglustat, or 20 mg/kg ATB200and 260 mg miglustat;

FIG. 26 is a graph showing the concentration-time profiles of miglustatin plasma in human subjects after dosing of 130 mg or 260 mg ofmiglustat;

FIG. 27 is a series of immunofluorescent micrographs of GAA and LAMP1levels in wild-type and Pompe fibroblasts;

FIG. 28 is a series of photomicrographs of muscle fibers from wild-typeand Gaa-knockout mice showing dystrophin, α- and β-dystroglycan, anddysferlin levels;

FIGS. 29A and 29B are a series of photomicrographs (200×) of musclefibers of rectus femoris (RF) and vastus lateralis/vastus medialis(VL/VM) from wild-type and Gaa-knockout mice treated with vehicle,alglucosidase alfa and ATB200 in the presence and absence of miglustat,showing LAMP1 IHC signals;

FIGS. 30A and 30B are a series of photomicrographs (200×) of musclefibers of RF and VL/VM from wild-type and Gaa-knockout mice treated withvehicle, alglucosidase alfa and ATB200 in the presence and absence ofmiglustat, showing LC3 II IHC signals;

FIGS. 31A and 31B are a series of photomicrographs (200×) muscle fibersof RF and VL/VM from wild-type and Gaa-knockout mice treated withvehicle, alglucosidase alfa and ATB200 in the presence and absence ofmiglustat, showing dysferlin IHC signals;

FIGS. 32A-32D are graphs showing glycogen levels in quadriceps, triceps,gastrocnemius and heart cells from wild-type and Gaa-knockout micetreated with vehicle, alglucosidase alfa and ATB200 in the presence andabsence of miglustat;

FIGS. 33A and 33B are graphs showing wire hand and grip strength muscledata for wild-type and Gaa-knockout mice treated with vehicle,alglucosidase alfa and ATB200 in the presence of miglustat;

FIGS. 34A-34G are graphs showing glycogen levels in quadriceps, tricepsand heart cells from wild-type and Gaa-knockout mice treated withvehicle, alglucosidase alfa and ATB200 in the presence and absence ofmiglustat;

FIG. 35 is a series of photomicrographs of muscle fibers of VL/VM fromwild-type and Gaa-knockout mice treated with vehicle, alglucosidase alfaand ATB200 in the presence and absence of miglustat, showing LAMP1, LC3and dysferlin IHC signals;

FIG. 36 is a graph showing the concentration-time profiles of GAAactivity in plasma in Gaa-knockout mice after administration of twobatches of ATB200 having different sialic acid content;

FIGS. 37A-37D are graphs showing glycogen levels in quadriceps, triceps,gastrocnemius and heart cells from wild-type and Gaa-knockout micetreated with vehicle, alglucosidase alfa and ATB200;

FIG. 38 is a graph showing alanine aminotransferase (ALT) levels inhuman patients after administration of ascending doses of ATB200 (5, 10and 20 mg/kg) followed by co-administration of ATB200 (20 mg/kg) andmiglustat (130 and 260 mg);

FIG. 39 is a graph showing aspartate aminotransferase (AST) levels inhuman patients after administration of ascending doses of ATB200 (5, 10and 20 mg/kg) followed by co-administration of ATB200 (20 mg/kg) andmiglustat (130 and 260 mg);

FIG. 40 is a graph showing creatine phosphokinase (CPK) levels in humanpatients after administration of ascending doses of ATB200 (5, 10 and 20mg/kg) followed by co-administration of ATB200 (20 mg/kg) and miglustat(130 and 260 mg);

FIG. 41 is a graph showing average ALT, AST and CPK levels in humanpatients after administration of ascending doses of ATB200 (5, 10 and 20mg/kg) followed by co-administration of ATB200 (20 mg/kg) and miglustat(130 and 260 mg); and

FIG. 42 is a series of photomicrographs (100× and 200×) of muscle fibersof vastus lateralis (VL) from wild-type and Gaa-knockout mice treatedwith vehicle, alglucosidase alfa and ATB200 in the presence and absenceof miglustat, showing dystrophin signals.

DEFINITIONS

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used.

Certain terms are discussed below, or elsewhere in the specification, toprovide additional guidance to the practitioner.

In the present specification, except where the context requiresotherwise due to express language or necessary implication, the word“comprises”, or variations such as “comprises” or “comprising” is usedin an inclusive sense i.e. to specify the presence of the statedfeatures but not to preclude the presence or addition of furtherfeatures in various embodiments of the invention.

As used herein, the term “Pompe disease,” also referred to as acidmaltase deficiency, glycogen storage disease type II (GSDII), andglycogenosis type II, is intended to refer to a genetic lysosomalstorage disorder characterized by mutations in the GAA gene, which codesfor the human acid α-glucosidase enzyme. The term includes but is notlimited to early and late onset forms of the disease, including but notlimited to infantile, juvenile and adult-onset Pompe disease.

As used herein, the term “acid α-glucosidase” is intended to refer to alysosomal enzyme which hydrolyzes α-1,4 linkages between the D-glucoseunits of glycogen, maltose, and isomaltose. Alternative names includebut are not limited to lysosomal α-glucosidase (EC:3.2.1.20);glucoamylase; 1,4-α-D-glucan glucohydrolase; amyloglucosidase;gamma-amylase and exo-1,4-α-glucosidase. Human acid α-glucosidase isencoded by the GAA gene (National Centre for Biotechnology Information(NCBI) Gene ID 2548), which has been mapped to the long arm ofchromosome 17 (location 17q25.2-q25.3). More than 500 mutations havecurrently been identified in the human GAA gene, many of which areassociated with Pompe disease. Mutations resulting in misfolding ormisprocessing of the acid α-glucosidase enzyme include T1064C(Leu355Pro) and C2104T (Arg702Cys). In addition, GAA mutations whichaffect maturation and processing of the enzyme include Leu405Pro andMet519Thr. The conserved hexapeptide WIDMNE at amino acid residues516-521 is required for activity of the acid α-glucosidase protein. Asused herein, the abbreviation “GAA” is intended to refer to the acidα-glucosidase enzyme, while the italicized abbreviation “GAA” isintended to refer to the human gene coding for the human acidα-glucosidase enzyme The italicized abbreviation “Gaa” is intended torefer to non-human genes coding for non-human acid α-glucosidaseenzymes, including but not limited to rat or mouse genes, and theabbreviation “Gaa” is intended to refer to non-human acid α-glucosidaseenzymes. Thus, the abbreviation “rhGAA” is intended to refer to therecombinant human acid α-glucosidase enzyme.

As used herein, the term “alglucosidase alfa” is intended to refer to arecombinant human acid α-glucosidase identified as[199-arginine,223-histidine]prepro-α-glucosidase (human); ChemicalAbstracts Registry Number 420794-05-0. Alglucosidase alfa is approvedfor marketing in the United States by Genzyme, as of Oct. 1, 2014, asthe products Lumizyme® and Myozyme®.

As used herein, the term “ATB200” is intended to refer to a recombinanthuman acid α-glucosidase described in co-pending patent applicationPCT/US2015/053252, the disclosure of which is herein incorporated byreference.

As used herein, the term “glycan” is intended to refer to apolysaccharide chain covalently bound to an amino acid residue on aprotein or polypeptide. As used herein, the term “N-glycan” or “N-linkedglycan” is intended to refer to a polysaccharide chain attached to anamino acid residue on a protein or polypeptide through covalent bindingto a nitrogen atom of the amino acid residue. For example, an N-glycancan be covalently bound to the side chain nitrogen atom of an asparagineresidue. Glycans can contain one or several monosaccharide units, andthe monosaccharide units can be covalently linked to form a straightchain or a branched chain. In at least one embodiment, N-glycan unitsattached to ATB200 can comprise one or more monosaccharide units eachindependently selected from N-acetylglucosamine, mannose, galactose orsialic acid. The N-glycan units on the protein can be determined by anyappropriate analytical technique, such as mass spectrometry. In someembodiments, the N-glycan units can be determined by liquidchromatography-tandem mass spectrometry (LC-MS/MS) utilizing aninstrument such as the Thermo Scientific Orbitrap Velos Pro™ MassSpectrometer, Thermo Scientific Orbitrap Fusion Lumos Tribid™ MassSpectrometer or Waters Xevo® G2-XS QTof Mass Spectrometer. As usedherein, the term “high-mannose N-glycan” is intended to refer to anN-glycan having one to six or more mannose units. In at least oneembodiment, a high mannose N-glycan unit can contain abis(N-acetylglucosamine) chain bonded to an asparagine residue andfurther bonded to a branched polymannose chain. As used hereininterchangeably, the term “M6P” or “mannose-6-phosphate” is intended torefer to a mannose unit phosphorylated at the 6 position; i.e. having aphosphate group bonded to the hydroxyl group at the 6 position. In atleast one embodiment, one or more mannose units of one or more N-glycanunits are phosphorylated at the 6 position to form mannose-6-phosphateunits. In at least one embodiment, the term “M6P” or“mannose-6-phosphate” refers to both a mannose phosphodiester havingN-acetylglucosamine (GlcNAc) as a “cap” on the phosphate group, as wellas a mannose unit having an exposed phosphate group lacking the GlcNAccap. In at least one embodiment, the N-glycans of a protein can havemultiple M6P groups, with at least one M6P group having a GlcNAc cap andat least one other M6P group lacking a GlcNAc cap.

As used herein, the term “complex N-glycan” is intended to refer to anN-glycan containing one or more galactose and/or sialic acid units. Inat least one embodiment, a complex N-glycan can be a high-mannoseN-glycan in which one or mannose units are further bonded to one or moremonosaccharide units each independently selected fromN-acetylglucosamine, galactose and sialic acid.

As used herein, the compound miglustat, also known asN-butyl-1-deoxynojirimycin or NB-DNJ or(2R,3R,4R,5S)-1-butyl-2-(hydroxymethyl)piperidine-3,4,5-triol, is acompound having the following chemical formula:

One formulation of miglustat is marketed commercially under the tradename Zavesca® as monotherapy for type 1 Gaucher disease.

As discussed below, pharmaceutically acceptable salts of miglustat mayalso be used in the present invention. When a salt of miglustat is used,the dosage of the salt will be adjusted so that the dose of miglustatreceived by the patient is equivalent to the amount which would havebeen received had the miglustat free base been used.

As used herein, the compound duvoglustat, also known as1-deoxynojirimycin or DNJ or(2R,3R,4R,5S)-2-(hydroxymethyl)piperidine-3,4,5-triol, is a compoundhaving the following chemical formula:

As used herein, the term “pharmacological chaperone” or sometimes simplythe term “chaperone” is intended to refer to a molecule thatspecifically binds to acid α-glucosidase and has one or more of thefollowing effects:

-   -   enhances the formation of a stable molecular conformation of the        protein;    -   enhances proper trafficking of the protein from the endoplasmic        reticulum to another cellular location, preferably a native        cellular location, so as to prevent endoplasmic        reticulum-associated degradation of the protein;    -   prevents aggregation of conformationally unstable or misfolded        proteins;    -   restores and/or enhances at least partial wild-type function,        stability, and/or activity of the protein; and/or    -   improves the phenotype or function of the cell harboring acid        α-glucosidase.

Thus, a pharmacological chaperone for acid α-glucosidase is a moleculethat binds to acid α-glucosidase, resulting in proper folding,trafficking, non-aggregation, and activity of acid α-glucosidase. Asused herein, this term includes but is not limited to activesite-specific chaperones (ASSCs) which bind in the active site of theenzyme, inhibitors or antagonists, and agonists. In at least oneembodiment, the pharmacological chaperone can be an inhibitor orantagonist of acid α-glucosidase. As used herein, the term “antagonist”is intended to refer to any molecule that binds to acid α-glucosidaseand either partially or completely blocks, inhibits, reduces, orneutralizes an activity of acid α-glucosidase. In at least oneembodiment, the pharmacological chaperone is miglustat. Anothernon-limiting example of a pharmacological chaperone for acidα-glucosidase is duvoglustat.

As used herein, the term “active site” is intended to refer to a regionof a protein that is associated with and necessary for a specificbiological activity of the protein. In at least one embodiment, theactive site can be a site that binds a substrate or other bindingpartner and contributes the amino acid residues that directlyparticipate in the making and breaking of chemical bonds. Active sitesin this invention can encompass catalytic sites of enzymes, antigenbinding sites of antibodies, ligand binding domains of receptors,binding domains of regulators, or receptor binding domains of secretedproteins. The active sites can also encompass transactivation,protein-protein interaction, or DNA binding domains of transcriptionfactors and regulators.

As used herein, the term “AUC” is intended to refer to a mathematicalcalculation to evaluate the body's total exposure over time to a givendrug. In a graph plotting how concentration in the blood of a drugadministered to a subject changes with time after dosing, the drugconcentration variable lies on the y-axis and time lies on the x-axis.The area between the drug concentration curve and the x-axis for adesignated time interval is the AUC (“area under the curve”). AUCs areused as a guide for dosing schedules and to compare the bioavailabilityof different drugs' availability in the body.

As used herein, the term “C_(max)” is intended to refer to the maximumplasma concentration of a drug achieved after administration to asubject.

As used herein, the term “volume of distribution” or “V” is intended torefer to the theoretical volume that would be necessary to contain thetotal amount of an administered drug at the same concentration that itis observed in the blood plasma, and represents the degree to which adrug is distributed in body tissue rather than the plasma. Higher valuesof V indicate a greater degree of tissue distribution. “Central volumeof distribution” or “V_(c)” is intended to refer to the volume ofdistribution within the blood and tissues highly perfused by blood.“Peripheral volume of distribution” or “V2” is intended to refer to thevolume of distribution within the peripheral tissue.

As used interchangeably herein, the terms “clearance”, “systemicclearance” or “CL” are intended to refer to the volume of plasma that iscompletely cleared of an administered drug per unit time. “Peripheralclearance” is intended to refer to the volume of peripheral tissue thatis cleared of an administered drug per unit time.

As used herein, the “therapeutically effective dose” and “effectiveamount” are intended to refer to an amount of acid α-glucosidase and/orof miglustat and/or of a combination thereof, which is sufficient toresult in a therapeutic response in a subject. A therapeutic responsemay be any response that a user (for example, a clinician) willrecognize as an effective response to the therapy, including anysurrogate clinical markers or symptoms described herein and known in theart. Thus, in at least one embodiment, a therapeutic response can be anamelioration or inhibition of one or more symptoms or markers of Pompedisease such as those known in the art. Symptoms or markers of Pompedisease include but are not limited to decreased acid α-glucosidasetissue activity; cardiomyopathy; cardiomegaly; progressive muscleweakness, especially in the trunk or lower limbs; profound hypotonia;macroglossia (and in some cases, protrusion of the tongue); difficultyswallowing, sucking, and/or feeding; respiratory insufficiency;hepatomegaly (moderate); laxity of facial muscles; areflexia; exerciseintolerance; exertional dyspnea; orthopnea; sleep apnea; morningheadaches; somnolence; lordosis and/or scoliosis; decreased deep tendonreflexes; lower back pain; and failure to meet developmental motormilestones. It should be noted that a concentration of miglustat thathas an inhibitory effect on acid α-glucosidase may constitute an“effective amount” for purposes of this invention because of dilution(and consequent shift in binding due to the change in equilibrium),bioavailability and metabolism of miglustat upon administration in vivo.

As used herein, the term “enzyme replacement therapy” or “ERT” isintended to refer to the introduction of a non-native, purified enzymeinto an individual having a deficiency in such enzyme. The administeredprotein can be obtained from natural sources or by recombinantexpression. The term also refers to the introduction of a purifiedenzyme in an individual otherwise requiring or benefiting fromadministration of a purified enzyme. In at least one embodiment, such anindividual suffers from enzyme insufficiency. The introduced enzyme maybe a purified, recombinant enzyme produced in vitro, or a proteinpurified from isolated tissue or fluid, such as, for example, placentaor animal milk, or from plants.

As used herein, the term “combination therapy” is intended to refer toany therapy wherein two or more individual therapies are administeredconcurrently or consecutively. In at least one embodiment, the resultsof the combination therapy are enhanced as compared to the effect ofeach therapy when it is performed individually. Enhancement may includeany improvement of the effect of the various therapies that may resultin an advantageous result as compared to the results achieved by thetherapies when performed alone. Enhanced effect or results can include asynergistic enhancement, wherein the enhanced effect is more than theadditive effects of each therapy when performed by itself; an additiveenhancement, wherein the enhanced effect is substantially equal to theadditive effect of each therapy when performed by itself; or less than asynergistic effect, wherein the enhanced effect is lower than theadditive effect of each therapy when performed by itself, but stillbetter than the effect of each therapy when performed by itself.Enhanced effect may be measured by any means known in the art by whichtreatment efficacy or outcome can be measured.

As used herein, the term “pharmaceutically acceptable” is intended torefer to molecular entities and compositions that are physiologicallytolerable and do not typically produce untoward reactions whenadministered to a human. Preferably, as used herein, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

As used herein, the term “carrier” is intended to refer to a diluent,adjuvant, excipient, or vehicle with which a compound is administered.Suitable pharmaceutical carriers are known in the art and, in at leastone embodiment, are described in “Remington's Pharmaceutical Sciences”by E. W. Martin, 18th Edition, or other editions.

As used herein, the terms “subject” or “patient” are intended to referto a human or non-human animal. In at least one embodiment, the subjectis a mammal. In at least one embodiment, the subject is a human.

As used herein, the term “anti-drug antibody” is intended to refer to anantibody specifically binding to a drug administered to a subject andgenerated by the subject as at least part of a humoral immune responseto administration of the drug to the subject. In at least one embodimentthe drug is a therapeutic protein drug product. The presence of theanti-drug antibody in the subject can cause immune responses rangingfrom mild to severe, including but not limited to life-threateningimmune responses which include but are not limited to anaphylaxis,cytokine release syndrome and cross-reactive neutralization ofendogenous proteins mediating critical functions. In addition oralternatively, the presence of the anti-drug antibody in the subject candecrease the efficacy of the drug.

As used herein, the term “neutralizing antibody” is intended to refer toan anti-drug antibody acting to neutralize the function of the drug. Inat least one embodiment, the therapeutic protein drug product is acounterpart of an endogenous protein for which expression is reduced orabsent in the subject. In at least one embodiment, the neutralizingantibody can act to neutralize the function of the endogenous protein.

As used herein, the terms “about” and “approximately” are intended torefer to an acceptable degree of error for the quantity measured giventhe nature or precision of the measurements. For example, the degree oferror can be indicated by the number of significant figures provided forthe measurement, as is understood in the art, and includes but is notlimited to a variation of ±1 in the most precise significant figurereported for the measurement. Typical exemplary degrees of error arewithin 20 percent (%), preferably within 10%, and more preferably within5% of a given value or range of values. Alternatively, and particularlyin biological systems, the terms “about” and “approximately” can meanvalues that are within an order of magnitude, preferably within 5-foldand more preferably within 2-fold of a given value. Numerical quantitiesgiven herein are approximate unless stated otherwise, meaning that theterm “about” or “approximately” can be inferred when not expresslystated.

The term “concurrently” as used herein is intended to mean at the sametime as or within a reasonably short period of time before or after, aswill be understood by those skilled in the art. For example, if twotreatments are administered concurrently with each other, one treatmentcan be administered before or after the other treatment, to allow fortime needed to prepare for the later of the two treatments. Therefore“concurrent administration” of two treatments includes but is notlimited to one treatment following the other by 20 minutes or less,about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes,about 2 minutes, about 1 minute or less than 1 minute.

The term “pharmaceutically acceptable salt” as used herein is intendedto mean a salt which is, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, generally water oroil-soluble or dispersible, and effective for their intended use. Theterm includes pharmaceutically-acceptable acid addition salts andpharmaceutically-acceptable base addition salts. Lists of suitable saltsare found in, for example, S. M. Birge et al., J. Pharm. Sci., 1977, 66,pp. 1-19, herein incorporated by reference.

The term “pharmaceutically-acceptable acid addition salt” as used hereinis intended to mean those salts which retain the biologicaleffectiveness and properties of the free bases and which are notbiologically or otherwise undesirable, formed with inorganic acidsincluding but not limited to hydrochloric acid, hydrobromic acid,sulfuric acid, sulfamic acid, nitric acid, phosphoric acid and the like,and organic acids including but not limited to acetic acid,trifluoroacetic acid, adipic acid, ascorbic acid, aspartic acid,benzenesulfonic acid, benzoic acid, butyric acid, camphoric acid,camphorsulfonic acid, cinnamic acid, citric acid, digluconic acid,ethanesulfonic acid, glutamic acid, glycolic acid, glycerophosphoricacid, hemisulfic acid, hexanoic acid, formic acid, fumaric acid,2-hydroxyethanesulfonic acid (isethionic acid), lactic acid,hydroxymaleic acid, malic acid, malonic acid, mandelic acid,mesitylenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid,nicotinic acid, 2-naphthalenesulfonic acid, oxalic acid, pamoic acid,pectinic acid, phenylacetic acid, 3-phenylpropionic acid, pivalic acid,propionic acid, pyruvic acid, salicylic acid, stearic acid, succinicacid, sulfanilic acid, tartaric acid, p-toluenesulfonic acid, undecanoicacid and the like.

The term “pharmaceutically-acceptable base addition salt” as used hereinis intended to mean those salts which retain the biologicaleffectiveness and properties of the free acids and which are notbiologically or otherwise undesirable, formed with inorganic basesincluding but not limited to ammonia or the hydroxide, carbonate, orbicarbonate of ammonium or a metal cation such as sodium, potassium,lithium, calcium, magnesium, iron, zinc, copper, manganese, aluminum andthe like. Salts derived from pharmaceutically-acceptable organicnontoxic bases include but are not limited to salts of primary,secondary, and tertiary amines, quaternary amine compounds, substitutedamines including naturally occurring substituted amines, cyclic aminesand basic ion-exchange resins, such as methylamine, dimethylamine,trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine,tripropylamine, tributylamine, ethanolamine, diethanolamine,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, hydrabamine, choline, betaine,ethylenediamine, glucosamine, methylglucamine, theobromine, purines,piperazine, piperidine, N-ethylpiperidine, tetramethylammoniumcompounds, tetraethylammonium compounds, pyridine, N,N-dimethylaniline,N-methylpiperidine, N-methylmorpholine, dicyclohexylamine,dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine,N,N′-dibenzylethylenediamine, polyamine resins and the like.

DETAILED DESCRIPTION

The present invention provides a method of treating Pompe disease in apatient in need thereof, the method including administering miglustat,or a pharmaceutically acceptable salt thereof, to the patient incombination with a recombinant human acid α-glucosidase, wherein therecombinant human acid α-glucosidase is expressed in Chinese hamsterovary (CHO) cells and comprises an increased content of N-glycan unitsbearing one or two mannose-6-phosphate residues when compared to acontent of N-glycan units bearing one or two mannose-6-phosphateresidues of alglucosidase alfa. In at least one embodiment, therecombinant human acid α-glucosidase has low levels of complex glycanswith terminal galactose. In another aspect, the present inventionprovides the use of miglustat and the recombinant human acidα-glucosidase in combination for the treatment of Pompe disease in apatient in need thereof.

In at least one embodiment, the miglustat is administered orally. In atleast one embodiment, the miglustat is administered at an oral dose ofabout 200 mg to about 600 mg, or at an oral dose of about 200 mg, about250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about500 mg, about 550 mg or about 600 mg. In at least one embodiment, themiglustat is administered at an oral dose of about 233 mg to about 400mg. In at least one embodiment, the miglustat is administered at an oraldose of about 250 to about 270 mg, or at an oral dose of about 250 mg,about 255 mg, about 260 mg, about 265 mg or about 270 mg. In at leastone embodiment, the miglustat is administered as an oral dose of about260 mg.

It will be understood by those skilled in the art that an oral dose ofmiglustat in the range of about 200 mg to 600 mg or any smaller rangetherewithin can be suitable for an adult patient with an average bodyweight of about 70 kg. For patients having a significantly lower bodyweight than about 70 kg, including but not limited to infants, childrenor underweight adults, a smaller dose may be considered suitable by aphysician.

Therefore, in at least one embodiment, the miglustat is administered asan oral dose of from about 50 mg to about 200 mg, or as an oral dose ofabout 50 mg, about 75 mg, about 100 mg, 125 mg, about 150 mg, about 175mg or about 200 mg. In at least one embodiment, the miglustat isadministered as an oral dose of from about 65 mg to about 195 mg, or asan oral dose of about 65 mg, about 130 mg or about 195 mg.

In at least one embodiment, the miglustat is administered as apharmaceutically acceptable dosage form suitable for oraladministration, and includes but is not limited to tablets, capsules,ovules, elixirs, solutions or suspensions, gels, syrups, mouth washes,or a dry powder for reconstitution with water or other suitable vehiclebefore use, optionally with flavoring and coloring agents forimmediate-, delayed-, modified-, sustained-, pulsed- orcontrolled-release applications. Solid compositions such as tablets,capsules, lozenges, pastilles, pills, boluses, powder, pastes, granules,bullets, dragees or premix preparations can also be used. In at leastone embodiment, the miglustat is administered as a tablet. In at leastone embodiment, the miglustat is administered as a capsule. In at leastone embodiment, the dosage form contains from about 50 mg to about 300mg of miglustat. In at least one embodiment, the dosage form containsabout 65 mg of miglustat. In at least one embodiment, the dosage formcontains about 130 mg of miglustat. In at least one embodiment, thedosage form contains about 260 mg of miglustat. It is contemplated thatwhen the dosage form contains about 65 mg of miglustat, the miglustatcan be administered as a dosage of four dosage forms, or a total dose of260 mg of miglustat. However, for patients who have a significantlylower weight than an average adult weight of 70 kg, including but notlimited to infants, children or underweight adults, the miglustat can beadministered as a dosage of one dosage form (a total dose of 65 mg ofmiglustat), two dosage forms (a total dose of 130 mg of miglustat), orthree dosage forms (a total dose of 195 mg of miglustat).

Solid and liquid compositions for oral use can be prepared according tomethods well known in the art. Such compositions can also contain one ormore pharmaceutically acceptable carriers and excipients which can be insolid or liquid form. Tablets or capsules can be prepared byconventional means with pharmaceutically acceptable excipients,including but not limited to binding agents, fillers, lubricants,disintegrants or wetting agents. Suitable pharmaceutically acceptableexcipients are known in the art and include but are not limited topregelatinized starch, polyvinylpyrrolidone, povidone, hydroxypropylmethylcellulose (HPMC), hydroxypropyl ethylcellulose (HPEC),hydroxypropyl cellulose (HPC), sucrose, gelatin, acacia, lactose,microcrystalline cellulose, calcium hydrogen phosphate, magnesiumstearate, stearic acid, glyceryl behenate, talc, silica, corn, potato ortapioca starch, sodium starch glycolate, sodium lauryl sulfate, sodiumcitrate, calcium carbonate, dibasic calcium phosphate, glycinecroscarmellose sodium and complex silicates. Tablets can be coated bymethods well known in the art. In at least one embodiment, the miglustatis administered as a formulation available commercially as Zavesca®(Actelion Pharmaceuticals).

In at least one embodiment, the recombinant human acid α-glucosidase isexpressed in Chinese hamster ovary (CHO) cells and comprises anincreased content of N-glycan units bearing one or moremannose-6-phosphate residues when compared to a content of N-glycanunits bearing one or more mannose-6-phosphate residues of alglucosidasealfa. In at least one embodiment, the acid α-glucosidase is arecombinant human acid α-glucosidase referred to herein as ATB200, asdescribed in co-pending international patent applicationPCT/US2015/053252. ATB200 has been shown to bind cation-independentmannose-6-phosphate receptors (CIMPR) with high affinity (K_(D)˜2-4 nM)and to be efficiently internalized by Pompe fibroblasts and skeletalmuscle myoblasts (K_(uptake)˜7-14 nM). ATB200 was characterized in vivoand shown to have a shorter apparent plasma half-life (t_(1/2)˜45 min)than alglucosidase alfa (t_(1/2)˜60 min).

In at least one embodiment, the recombinant human acid α-glucosidase isan enzyme having an amino acid sequence as set forth in SEQ ID NO: 1,SEQ ID NO: 2 (or as encoded by SEQ ID NO: 2), SEQ ID NO: 3, SEQ ID NO: 4or SEQ ID NO: 5.

SEQ ID NO: 1 Met Gly Val Arg His Pro Pro Cys Ser His Arg LeuLeu Ala Val Cys Ala Leu Val Ser Leu Ala Thr AlaAla Leu Leu Gly His Ile Leu Leu His Asp Phe LeuLeu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro ValLeu Glu Glu Thr His Pro Ala His Gln Gln Gly AlaSer Arg Pro Gly Pro Arg Asp Ala Gln Ala His ProGly Arg Pro Arg Ala Val Pro Thr Gln Cys Asp ValPro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp LysAla Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly CysCys Tyr Ile Pro Ala Lys Gln Gly Leu Gln Gly AlaGln Met Gly Gln Pro Trp Cys Phe Phe Pro Pro SerTyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser SerGlu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr ThrPro Thr Phe Phe Pro Lys Asp Ile Leu Thr Leu ArgLeu Asp Val Met Met Glu Thr Glu Asn Arg Leu HisPhe Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr GluVal Pro Leu Glu Thr Pro Arg Val His Ser Arg AlaPro Ser Pro Leu Tyr Ser Val Glu Phe Ser Glu GluPro Phe Gly Val Ile Val His Arg Gln Leu Asp GlyArg Val Leu Leu Asn Thr Thr Val Ala Pro Leu PhePhe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser LeuPro Ser Gln Tyr Ile Thr Gly Leu Ala Glu His LeuSer Pro Leu Met Leu Ser Thr Ser Trp Thr Arg IleThr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro GlyAla Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu AlaLeu Glu Asp Gly Gly Ser Ala His Gly Val Phe LeuLeu Asn Ser Asn Ala Met Asp Val Val Leu Gln ProSer Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly IleLeu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro LysSer Val Val Gln Gln Tyr Leu Asp Val Val Gly TyrPro Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe HisLeu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile ThrArg Gln Val Val Glu Asn Met Thr Arg Ala His PhePro Leu Asp Val Gln Trp Asn Asp Leu Asp Tyr MetAsp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp GlyPhe Arg Asp Phe Pro Ala Met Val Gln Glu Leu HisGln Gly Gly Arg Arg Tyr Met Met Ile Val Asp ProAla Ile Ser Ser Ser Gly Pro Ala Gly Ser Tyr ArgPro Tyr Asp Glu Gly Leu Arg Arg Gly Val Phe IleThr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys ValTrp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr AsnPro Thr Ala Leu Ala Trp Trp Glu Asp Met Val AlaGlu Phe His Asp Gln Val Pro Phe Asp Gly Met TrpIle Asp Met Asn Glu Pro Ser Asn Phe Ile Arg GlySer Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu AsnPro Pro Tyr Val Pro Gly Val Val Gly Gly Thr LeuGln Ala Ala Thr Ile Cys Ala Ser Ser His Gln PheLeu Ser Thr His Tyr Asn Leu His Asn Leu Tyr GlyLeu Thr Glu Ala Ile Ala Ser His Arg Ala Leu ValLys Ala Arg Gly Thr Arg Pro Phe Val Ile Ser ArgSer Thr Phe Ala Gly His Gly Arg Tyr Ala Gly HisTrp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln LeuAla Ser Ser Val Pro Glu Ile Leu Gln Phe Asn LeuLeu Gly Val Pro Leu Val Gly Ala Asp Val Cys GlyPhe Leu Gly Asn Thr Ser Glu Glu Leu Cys Val ArgTrp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met ArgAsn His Asn Ser Leu Leu Ser Leu Pro Gln Glu ProTyr Ser Phe Ser Glu Pro Ala Gln Gln Ala Met ArgLys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro HisLeu Tyr Thr Leu Phe His Gln Ala His Val Ala GlyGlu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe ProLys Asp Ser Ser Thr Trp Thr Val Asp His Gln LeuLeu Trp Gly Glu Ala Leu Leu Ile Thr Pro Val LeuGln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe ProLeu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro IleGlu Ala Leu Gly Ser Leu Pro Pro Pro Pro Ala AlaPro Arg Glu Pro Ala Ile His Ser Glu Gly Gln TrpVal Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn ValHis Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln GlyPro Gly Leu Thr Thr Thr Glu Ser Arg Gln Gln ProMet Ala Leu Ala Val Ala Leu Thr Lys Gly Gly GluAla Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu SerLeu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln ValIle Phe Leu Ala Arg Asn Asn Thr Ile Val Asn GluLeu Val Arg Val Thr Ser Glu Gly Ala Gly Leu GlnLeu Gln Lys Val Thr Val Leu Gly Val Ala Thr AlaPro Gln Gln Val Leu Ser Asn Gly Val Pro Val SerAsn Phe Thr Tyr Ser Pro Asp Thr Lys Val Leu AspIle Cys Val Ser Leu Leu Met Gly Glu Gln Phe Leu Val Ser Trp CysSEQ ID NO: 2 cagttgggaaagctgaggttgtcgccggggccgcgggtggaggtcggggatgaggcagcaggtaggacagtgacctcggtgacgcgaaggaccccggccacctctaggttctcctcgtccgcccgttgttcagcgagggaggctctgggcctgccgcagctgacggggaaactgaggcacggagcgggcctgtaggagctgtccaggccatctccaaccatgggagtgaggcacccgccctgctcccaccggctcctggccgtctgcgccctcgtgtccttggcaaccgctgcactcctggggcacatcctactccatgatttcctgctggttccccgagagctgagtggctcctccccagtcctggaggagactcacccagctcaccagcagggagccagcagaccagggccccgggatgcccaggcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccctggtgcttcttcccacccagctaccccagctacaagctggagaacctgagctcctctgaaatgggctacacggccaccctgacccgtaccacccccaccttcttccccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttctccgaggagcccttcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgttctttgcggaccagttccttcagctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcaccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgcccttagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctacgacgagggtctgcggaggggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagccttccaacttcatcagaggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggttggggggaccctccaggcggccaccatctgtgcctccagccaccagtttctctccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagctgtgtgtgcgctggacccagctgggggccttctaccccttcatgcggaaccacaacagcctgctcagtctgccccaggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactcctcccccacctctacacactgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaaggactctagcacctggactgtggaccaccagctcctgtggggggaggccctgctcatcaccccagtgctccaggccgggaaggccgaagtgactggctacttccccttgggcacatggtacgacctgcagacggtgccaatagaggcccttggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgccagcagcccatggccctggctgtggccctgaccaagggtggagaggcccgaggggagctgttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcgtcagctggtgttagccgggcggagtgtgttagtctctccagagggaggctggttccccagggaagcagagcctgtgtgcgggcagcagctgtgtgcgggcctgggggttgcatgtgtcacctggagctgggcactaaccattccaagccgccgcatcgcttgtttccacctcctgggccggggctctggcccccaacgtgtctaggagagctttctccctagatcgcactgtgggccggggcctggagggctgctctgtgttaataagattgtaaggtttgccctcctcacctgttgccggcatgcgggtagtattagccacccccctccatctgttcccagcaccggagaagggggtgctcaggtggaggtgtggggtatgcacctgagctcctgcttcgcgcctgctgctctgccccaacgcgaccgcttcccggctgcccagagggctggatgcctgccggtccccgagcaagcctgggaactcaggaaaattcacaggacttgggagattctaaatcttaagtgcaattattttaataaaaggggcatttggaatc SEQ ID NO: 3Met Gly Val Arg His Pro Pro Cys Ser His Arg LeuLeu Ala Val Cys Ala Leu Val Ser Leu Ala Thr AlaAla Leu Leu Gly His Ile Leu Leu His Asp Phe LeuLeu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro ValLeu Glu Glu Thr His Pro Ala His Gln Gln Gly AlaSer Arg Pro Gly Pro Arg Asp Ala Gln Ala His ProGly Arg Pro Arg Ala Val Pro Thr Gln Cys Asp ValPro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp LysAla Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly CysCys Tyr Ile Pro Ala Lys Gln Gly Leu Gln Gly AlaGln Met Gly Gln Pro Trp Cys Phe Phe Pro Pro SerTyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser SerGlu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr ThrPro Thr Phe Phe Pro Lys Asp Ile Leu Thr Leu ArgLeu Asp Val Met Met Glu Thr Glu Asn Arg Leu HisPhe Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr GluVal Pro Leu Glu Thr Pro Arg Val His Ser Arg AlaPro Ser Pro Leu Tyr Ser Val Glu Phe Ser Glu GluPro Phe Gly Val Ile Val His Arg Gln Leu Asp GlyArg Val Leu Leu Asn Thr Thr Val Ala Pro Leu PhePhe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser LeuPro Ser Gln Tyr Ile Thr Gly Leu Ala Glu His LeuSer Pro Leu Met Leu Ser Thr Ser Trp Thr Arg IleThr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro GlyAla Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu AlaLeu Glu Asp Gly Gly Ser Ala His Gly Val Phe LeuLeu Asn Ser Asn Ala Met Asp Val Val Leu Gln ProSer Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly IleLeu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro LysSer Val Val Gln Gln Tyr Leu Asp Val Val Gly TyrPro Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe HisLeu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile ThrArg Gln Val Val Glu Asn Met Thr Arg Ala His PhePro Leu Asp Val Gln Trp Asn Asp Leu Asp Tyr MetAsp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp GlyPhe Arg Asp Phe Pro Ala Met Val Gln Glu Leu HisGln Gly Gly Arg Arg Tyr Met Met Ile Val Asp ProAla Ile Ser Ser Ser Gly Pro Ala Gly Ser Tyr ArgPro Tyr Asp Glu Gly Leu Arg Arg Gly Val Phe IleThr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys ValTrp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr AsnPro Thr Ala Leu Ala Trp Trp Glu Asp Met Val AlaGlu Phe His Asp Gln Val Pro Phe Asp Gly Met TrpIle Asp Met Asn Glu Pro Ser Asn Phe Ile Arg GlySer Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu AsnPro Pro Tyr Val Pro Gly Val Val Gly Gly Thr LeuGln Ala Ala Thr Ile Cys Ala Ser Ser His Gln PheLeu Ser Thr His Tyr Asn Leu His Asn Leu Tyr GlyLeu Thr Glu Ala Ile Ala Ser His Arg Ala Leu ValLys Ala Arg Gly Thr Arg Pro Phe Val Ile Ser ArgSer Thr Phe Ala Gly His Gly Arg Tyr Ala Gly HisTrp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln LeuAla Ser Ser Val Pro Glu Ile Leu Gln Phe Asn LeuLeu Gly Val Pro Leu Val Gly Ala Asp Val Cys GlyPhe Leu Gly Asn Thr Ser Glu Glu Leu Cys Val ArgTrp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met ArgAsn His Asn Ser Leu Leu Ser Leu Pro Gln Glu ProTyr Ser Phe Ser Glu Pro Ala Gln Gln Ala Met ArgLys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro HisLeu Tyr Thr Leu Phe His Gln Ala His Val Ala GlyGlu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe ProLys Asp Ser Ser Thr Trp Thr Val Asp His Gln LeuLeu Trp Gly Glu Ala Leu Leu Ile Thr Pro Val LeuGln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe ProLeu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro IleGlu Ala Leu Gly Ser Leu Pro Pro Pro Pro Ala AlaPro Arg Glu Pro Ala Ile His Ser Glu Gly Gln TrpVal Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn ValHis Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln GlyPro Gly Leu Thr Thr Thr Glu Ser Arg Gln Gln ProMet Ala Leu Ala Val Ala Leu Thr Lys Gly Gly GluAla Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu SerLeu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln ValIle Phe Leu Ala Arg Asn Asn Thr Ile Val Asn GluLeu Val Arg Val Thr Ser Glu Gly Ala Gly Leu GlnLeu Gln Lys Val Thr Val Leu Gly Val Ala Thr AlaPro Gln Gln Val Leu Ser Asn Gly Val Pro Val SerAsn Phe Thr Tyr Ser Pro Asp Thr Lys Val Leu AspIle Cys Val Ser Leu Leu Met Gly Glu Gln Phe LeuVal Ser Trp Cys Met Gly Val Arg His Pro Pro CysSer His Arg Leu Leu Ala Val Cys SEQ ID NO: 4Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu GlyHis Ile Leu Leu His Asp Phe Leu Leu Val Pro ArgGlu Leu Ser Gly Ser Ser Pro Val Leu Glu Glu ThrHis Pro Ala His Gln Gln Gly Ala Ser Arg Pro GlyPro Arg Asp Ala Gln Ala His Pro Gly Arg Pro ArgAla Val Pro Thr Gln Cys Asp Val Pro Pro Asn SerArg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr GlnGlu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile ProAla Lys Gln Gly Leu Gln Gly Ala Gln Met Gly GlnPro Trp Cys Phe Phe Pro Pro Ser Tyr Pro Ser TyrLys Leu Glu Asn Leu Ser Ser Ser Glu Met Gly TyrThr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe PhePro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val MetMet Glu Thr Glu Asn Arg Leu His Phe Thr Ile LysAsp Pro Ala Asn Arg Arg Tyr Glu Val Pro Leu GluThr Pro His Val His Ser Arg Ala Pro Ser Pro LeuTyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly ValIle Val Arg Arg Gln Leu Asp Gly Arg Val Leu LeuAsn Thr Thr Val Ala Pro Leu Phe Phe Ala Asp GlnPhe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln TyrIle Thr Gly Leu Ala Glu His Leu Ser Pro Leu MetLeu Ser Thr Ser Trp Thr Arg Ile Thr Leu Trp AsnArg Asp Leu Ala Pro Thr Pro Gly Ala Asn Leu TyrGly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp GlyGly Ser Ala His Gly Val Phe Leu Leu Asn Ser AsnAla Met Asp Val Val Leu Gln Pro Ser Pro Ala LeuSer Trp Arg Ser Thr Gly Gly Ile Leu Asp Val TyrIle Phe Leu Gly Pro Glu Pro Lys Ser Val Val GlnGln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met ProPro Tyr Trp Gly Leu Gly Phe His Leu Cys Arg TrpGly Tyr Ser Ser Thr Ala Ile Thr Arg Gln Val ValGlu Asn Met Thr Arg Ala His Phe Pro Leu Asp ValGln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg ArgAsp Phe Thr Phe Asn Lys Asp Gly Phe Arg Asp PhePro Ala Met Val Gln Glu Leu His Gln Gly Gly ArgArg Tyr Met Met Ile Val Asp Pro Ala Ile Ser SerSer Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp GluGly Leu Arg Arg Gly Val Phe Ile Thr Asn Glu ThrGly Gln Pro Leu Ile Gly Lys Val Trp Pro Gly SerThr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala LeuAla Trp Trp Glu Asp Met Val Ala Glu Phe His AspGln Val Pro Phe Asp Gly Met Trp Ile Asp Met AsnGlu Pro Ser Asn Phe Ile Arg Gly Ser Glu Asp GlyCys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr ValPro Gly Val Val Gly Gly Thr Leu Gln Ala Ala ThrIle Cys Ala Ser Ser His Gln Phe Leu Ser Thr HisTyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu AlaIle Ala Ser His Arg Ala Leu Val Lys Ala Arg GlyThr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe AlaGly His Gly Arg Tyr Ala Gly His Trp Thr Gly AspVal Trp Ser Ser Trp Glu Gln Leu Ala Ser Ser ValPro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val ProLeu Val Gly Ala Asp Val Cys Gly Phe Leu Gly AsnThr Ser Glu Glu Leu Cys Val Arg Trp Thr Gln LeuGly Ala Phe Tyr Pro Phe Met Arg Asn His Asn SerLeu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe SerGlu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu ThrLeu Arg Tyr Ala Leu Leu Pro His Leu Tyr Thr LeuPhe His Gln Ala His Val Ala Gly Glu Thr Val AlaArg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser SerThr Trp Thr Val Asp His Gln Leu Leu Trp Gly GluAla Leu Leu Ile Thr Pro Val Leu Gln Ala Gly LysAla Glu Val Thr Gly Tyr Phe Pro Leu Gly Thr TrpTyr Asp Leu Gln Thr Val Pro Val Glu Ala Leu GlySer Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu ProAla Ile His Ser Glu Gly Gln Trp Val Thr Leu ProAla Pro Leu Asp Thr Ile Asn Val His Leu Arg AlaGly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu ThrThr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu AlaVal Ala Leu Thr Lys Gly Gly Glu Ala Arg Gly GluLeu Phe Trp Asp Asp Gly Glu Ser Leu Glu Val LeuGlu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu AlaArg Asn Asn Thr Ile Val Asn Glu Leu Val Arg ValThr Ser Glu Gly Ala Gly Leu Gln Leu Gln Lys ValThr Val Leu Gly Val Ala Thr Ala Pro Gln Gln ValLeu Ser Asn Gly Val Pro Val Ser Asn Phe Thr TyrSer Pro Asp Thr Lys Val Leu Asp Ile Cys Val SerLeu Leu Met Gly Glu Gln Phe Leu Val Ser Trp Cys SEQ ID NO: 5Gln Gln Gly Ala Ser Arg Pro Gly Pro Arg Asp AlaGln Ala His Pro Gly Arg Pro Arg Ala Val Pro ThrGln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp CysAla Pro Asp Lys Ala Ile Thr Gln Glu Gln Cys GluAla Arg Gly Cys Cys Tyr Ile Pro Ala Lys Gln GlyLeu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys PhePhe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu AsnLeu Ser Ser Ser Glu Met Gly Tyr Thr Ala Thr LeuThr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp IleLeu Thr Leu Arg Leu Asp Val Met Met Glu Thr GluAsn Arg Leu His Phe Thr Ile Lys Asp Pro Ala AsnArg Arg Tyr Glu Val Pro Leu Glu Thr Pro Arg ValHis Ser Arg Ala Pro Ser Pro Leu Tyr Ser Val GluPhe Ser Glu Glu Pro Phe Gly Val Ile Val His ArgGln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr ValAla Pro Leu Phe Phe Ala Asp Gln Phe Leu Gln LeuSer Thr Ser Leu Pro Ser Gln Tyr Ile Thr Gly LeuAla Glu His Leu Ser Pro Leu Met Leu Ser Thr SerTrp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu AlaPro Thr Pro Gly Ala Asn Leu Tyr Gly Ser His ProPhe Tyr Leu Ala Leu Glu Asp Gly Gly Ser Ala HisGly Val Phe Leu Leu Asn Ser Asn Ala Met Asp ValVal Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg SerThr Gly Gly Ile Leu Asp Val Tyr Ile Phe Leu GlyPro Glu Pro Lys Ser Val Val Gln Gln Tyr Leu AspVal Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp GlyLeu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser SerThr Ala Ile Thr Arg Gln Val Val Glu Asn Met ThrArg Ala His Phe Pro Leu Asp Val Gln Trp Asn AspLeu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr PheAsn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met ValGln Glu Leu His Gln Gly Gly Arg Arg Tyr Met MetIle Val Asp Pro Ala Ile Ser Ser Ser Gly Pro AlaGly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg ArgGly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro LeuIle Gly Lys Val Trp Pro Gly Ser Thr Ala Phe ProAsp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp GluAsp Met Val Ala Glu Phe His Asp Gln Val Pro PheAsp Gly Met Trp Ile Asp Met Asn Glu Pro Ser AsnPhe Ile Arg Gly Ser Glu Asp Gly Cys Pro Asn AsnGlu Leu Glu Asn Pro Pro Tyr Val Pro Gly Val ValGly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala SerSer His Gln Phe Leu Ser Thr His Tyr Asn Leu HisAsn Leu Tyr Gly Leu Thr Glu Ala Ile Ala Ser HisArg Ala Leu Val Lys Ala Arg Gly Thr Arg Pro PheVal Ile Ser Arg Ser Thr Phe Ala Gly His Gly ArgTyr Ala Gly His Trp Thr Gly Asp Val Trp Ser SerTrp Glu Gln Leu Ala Ser Ser Val Pro Glu Ile LeuGln Phe Asn Leu Leu Gly Val Pro Leu Val Gly AlaAsp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu GluLeu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe TyrPro Phe Met Arg Asn His Asn Ser Leu Leu Ser LeuPro Gln Glu Pro Tyr Ser Phe Ser Glu Pro Ala GlnGln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr AlaLeu Leu Pro His Leu Tyr Thr Leu Phe His Gln AlaHis Val Ala Gly Glu Thr Val Ala Arg Pro Leu PheLeu Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr ValAsp His Gln Leu Leu Trp Gly Glu Ala Leu Leu IleThr Pro Val Leu Gln Ala Gly Lys Ala Glu Val ThrGly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu GlnThr Val Pro Ile Glu Ala Leu Gly Ser Leu Pro ProPro Pro Ala Ala Pro Arg Glu Pro Ala Ile His SerGlu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu AspThr Ile Asn Val His Leu Arg Ala Gly Tyr Ile IlePro Leu Gln Gly Pro Gly Leu Thr Thr Thr Glu SerArg Gln Gln Pro Met Ala Leu Ala Val Ala Leu ThrLys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp AspAsp Gly Glu Ser Leu Glu Val Leu Glu Arg Gly AlaTyr Thr Gln Val Ile Phe Leu Ala Arg Asn Asn ThrIle Val Asn Glu Leu Val Arg Val Thr Ser Glu GlyAla Gly Leu Gln Leu Gln Lys Val Thr Val Leu GlyVal Ala Thr Ala Pro Gln Gln Val Leu Ser Asn GlyVal Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp ThrLys Val Leu Asp Ile Cys Val Ser Leu Leu Met GlyGlu Gln Phe Leu Val Ser Trp Cys

In at least one embodiment, the recombinant human acid α-glucosidase hasa wild-type GAA amino acid sequence as set forth in SEQ ID NO: 1, asdescribed in U.S. Pat. No. 8,592,362 and has GenBank accession numberAHE24104.1 (GI:568760974). In at least one embodiment, the recombinanthuman acid α-glucosidase has a wild-type GAA amino acid sequence asencoded in SEQ ID NO: 2, the mRNA sequence having GenBank accessionnumber Y00839.1. In at least one embodiment, the recombinant human acidα-glucosidase has a wild-type GAA amino acid sequence as set forth inSEQ ID NO: 3. In at least one embodiment, the recombinant human acidα-glucosidase has a GAA amino acid sequence as set forth in SEQ ID NO:4, and has National Center for Biotechnology Information (NCBI)accession number NP_000143.2. In at least one embodiment, therecombinant human acid α-glucosidase is glucosidase alfa, the human acidα-glucosidase enzyme encoded by the most predominant of nine observedhaplotypes of the GAA gene.

In at least one embodiment, the recombinant human acid α-glucosidase isinitially expressed as having the full-length 952 amino acid sequence ofwild-type GAA as set forth in SEQ ID NO: 1, and the recombinant humanacid α-glucosidase undergoes intracellular processing that removes aportion of the amino acids, e.g. the first 56 amino acids. Accordingly,the recombinant human acid α-glucosidase that is secreted by the hostcell can have a shorter amino acid sequence than the recombinant humanacid α-glucosidase that is initially expressed within the cell. In atleast one embodiment, the shorter protein can have the amino acidsequence set forth in SEQ ID NO: 5, which only differs from SEQ ID NO: 1in that the first 56 amino acids comprising the signal peptide andprecursor peptide have been removed, thus resulting in a protein having896 amino acids. Other variations in the number of amino acids is alsopossible, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15 or more deletions, substitutions and/or insertions relative to theamino acid sequence described by SEQ ID NO: 1 or SEQ ID NO: 5. In someembodiments, the rhGAA product includes a mixture of recombinant humanacid α-glucosidase molecules having different amino acid lengths.

In at least one embodiment, the recombinant human acid α-glucosidaseundergoes post-translational and/or chemical modifications at one ormore amino acid residues in the protein. For example, methionine andtryptophan residues can undergo oxidation. As another example, theN-terminal glutamine can form pyro-glutamate. As another example,asparagine residues can undergo deamidation to aspartic acid. As yetanother example, aspartic acid residues can undergo isomerization toiso-aspartic acid. As yet another example, unpaired cysteine residues inthe protein can form disulfide bonds with free glutathione and/orcysteine. Accordingly, in some embodiments the enzyme is initiallyexpressed as having an amino acid sequence as set forth in SEQ ID NO: 1,SEQ ID NO: 2 (or as encoded by SEQ ID NO: 2), SEQ ID NO: 3, SEQ ID NO: 4or SEQ ID NO: 5, and the enzyme undergoes one or more of thesepost-translational and/or chemical modifications. Such modifications arealso within the scope of the present disclosure.

Polynucleotide sequences encoding GAA and such variant human GAAs arealso contemplated and may be used to recombinantly express rhGAAsaccording to the invention.

Preferably, no more than 70, 65, 60, 55, 45, 40, 35, 30, 25, 20, 15, 10,or 5% of the total recombinant human acid α-glucosidase molecules lackan N-glycan unit bearing one or more mannose-6-phosphate residues orlacks a capacity to bind to the cation independent mannose-6-phosphatereceptor (CIMPR). Alternatively, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 99%, <100% or more of the recombinant human acidα-glucosidase molecules comprise at least one N-glycan unit bearing oneor more mannose-6-phosphate residues or has the capacity to bind toCIMPR.

The recombinant human acid α-glucosidase molecules may have 1, 2, 3 or 4mannose-6-phosphate (M6P) groups on their glycans. For example, only oneN-glycan on a recombinant human acid α-glucosidase molecule may bear M6P(mono-phosphorylated), a single N-glycan may bear two M6P groups(bis-phosphorylated), or two different N-glycans on the same recombinanthuman acid α-glucosidase molecule may each bear single M6P groups.Recombinant human acid α-glucosidase molecules may also have N-glycansbearing no M6P groups. In another embodiment, on average the N-glycanscontain greater than 2.5 mol/mol of M6P and greater than 4 mol/molsialic acid, such that the recombinant human acid α-glucosidasecomprises on average at least 2.5 moles of mannose-6-phosphate residuesper mole of recombinant human acid α-glucosidase and at least 4 moles ofsialic acid per mole of recombinant human acid α-glucosidase. On averageat least about 3, 4, 5, 6, 7, 8, 9, or 10% of the total glycans on therecombinant human acid α-glucosidase may be in the form of a mono-M6Pglycan, for example, about 6.25% of the total glycans may carry a singleM6P group and on average, at least about 0.5, 1, 1.5, 2.0, 2.5, 3.0% ofthe total glycans on the recombinant human acid α-glucosidase are in theform of a bis-M6P glycan and on average less than 25% of totalrecombinant human acid α-glucosidase contains no phosphorylated glycanbinding to CIMPR.

The recombinant human acid α-glucosidase may have an average content ofN-glycans carrying M6P ranging from 0.5 to 7.0 mol/mol recombinant humanacid α-glucosidase or any intermediate value of subrange including 0.5,1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0mol/mol recombinant human acid α-glucosidase. The recombinant human acidα-glucosidase can be fractionated to provide recombinant human acidα-glucosidase preparations with different average numbers of M6P-bearingor bis-M6P-bearing glycans thus permitting further customization ofrecombinant human acid α-glucosidase targeting to the lysosomes intarget tissues by selecting a particular fraction or by selectivelycombining different fractions.

Up to 60% of the N-glycans on the recombinant human acid α-glucosidasemay be fully sialylated, for example, up to 10%, 20%, 30%, 40%, 50% or60% of the N-glycans may be fully sialylated. In some embodiments from 4to 20% of the total N-glycans are fully sialylated. In other embodimentsno more than 5%, 10%, 20% or 30% of N-glycans on the recombinant humanacid α-glucosidase carry sialic acid and a terminal galactose residue(Gal). This range includes all intermediate values and subranges, forexample, 7 to 30% of the total N-glycans on the recombinant human acidα-glucosidase can carry sialic acid and terminal galactose. In yet otherembodiments, no more than 5, 10, 15, 16, 17, 18, 19 or 20% of theN-glycans on the recombinant human acid α-glucosidase have a terminalgalactose only and do not contain sialic acid. This range includes allintermediate values and subranges, for example, from 8 to 19% of thetotal N-glycans on the recombinant human acid α-glucosidase in thecomposition may have terminal galactose only and do not contain sialicacid.

In other embodiments of the invention, 40, 45, 50, 55 to 60% of thetotal N-glycans on the recombinant human acid α-glucosidase are complextype N-glycans; or no more than 1, 2, 3, 4, 5, 6, 7% of total N-glycanson the recombinant human acid α-glucosidase are hybrid-type N-glycans;no more than 5, 10, or 15% of the high mannose-type N-glycans on therecombinant human acid α-glucosidase are non-phosphorylated; at least 5%or 10% of the high mannose-type N-glycans on the recombinant human acidα-glucosidase are mono-M6P phosphorylated; and/or at least 1 or 2% ofthe high mannose-type N-glycans on the recombinant human acidα-glucosidase are bis-M6P phosphorylated. These values include allintermediate values and subranges. A recombinant human acidα-glucosidase may meet one or more of the content ranges describedabove.

In some embodiments, the recombinant human acid α-glucosidase will bear,on average, 2.0 to 8.0 moles of sialic acid residues per mole ofrecombinant human acid α-glucosidase. This range includes allintermediate values and subranges including 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 mol residues/mol recombinanthuman acid α-glucosidase. Without being bound by theory, it is believedthat the presence of N-glycan units bearing sialic acid residues mayprevent non-productive clearance of the recombinant human acidα-glucosidase by asialoglycoprotein receptors.

In one or more embodiments, the rhGAA has M6P and/or sialic acid unitsat certain N-glycosylation sites of the recombinant human lysosomalprotein. For example, there are seven potential N-linked glycosylationsites on rhGAA. These potential glycosylation sites are at the followingpositions of SEQ ID NO: 5: N84, N177, N334, N414, N596, N826 and N869.Similarly, for the full-length amino acid sequence of SEQ ID NO: 1,these potential glycosylation sites are at the following positions:N140, N233, N390, N470, N652, N882 and N925. Other variants of rhGAA canhave similar glycosylation sites, depending on the location ofasparagine residues. Generally, sequences of ASN-X-SER or ASN-X-THR inthe protein amino acid sequence indicate potential glycosylation sites,with the exception that X cannot be HIS or PRO.

In various embodiments, the rhGAA has a certain N-glycosylation profile.In one or more embodiments, at least 20% of the rhGAA is phosphorylatedat the first N-glycosylation site (e.g. N84 for SEQ ID NO: 5 and N140for SEQ ID NO: 1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA can bephosphorylated at the first N-glycosylation site. This phosphorylationcan be the result of mono-M6P and/or bis-M6P units. In some embodiments,at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or 95% of the rhGAA bears a mono-M6P unit at thefirst N-glycosylation site. In some embodiments, at least 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or95% of the rhGAA bears a bis-M6P unit at the first N-glycosylation site.

In one or more embodiments, at least 20% of the rhGAA is phosphorylatedat the second N-glycosylation site (e.g. N177 for SEQ ID NO: 5 and N223for SEQ ID NO: 1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA can bephosphorylated at the second N-glycosylation site. This phosphorylationcan be the result of mono-M6P and/or bis-M6P units. In some embodiments,at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or 95% of the rhGAA bears a mono-M6P unit at thesecond N-glycosylation site. In some embodiments, at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90% or 95% of the rhGAA bears a bis-M6P unit at the secondN-glycosylation site. In one or more embodiments, at least 5% of therhGAA is phosphorylated at the third N-glycosylation site (e.g. N334 forSEQ ID NO: 5 and N390 for SEQ ID NO: 1). In other embodiments, less than5%, 10%, 15%, 20% or 25% of the rhGAA is phosphorylated at the thirdN-glycosylation site. For example, the third N-glycosylation site canhave a mixture of non-phosphorylated high mannose glycans, di-, tri-,and tetra-antennary complex glycans, and hybrid glycans as the majorspecies. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45% or 50% of the rhGAA is sialylated at the thirdN-glycosylation site.

In one or more embodiments, at least 20% of the rhGAA is phosphorylatedat the fourth N-glycosylation site (e.g. N414 for SEQ ID NO: 5 and N470for SEQ ID NO: 1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA can bephosphorylated at the fourth N-glycosylation site. This phosphorylationcan be the result of mono-M6P and/or bis-M6P units. In some embodiments,at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or 95% of the rhGAA bears a mono-M6P unit at thefourth N-glycosylation site. In some embodiments, at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90% or 95% of the rhGAA bears a bis-M6P unit at the fourthN-glycosylation site. In some embodiments, at least 3%, 5%, 8%, 10%,15%, 20% or 25% of the rhGAA is sialylated at the fourth N-glycosylationsite.

In one or more embodiments, at least 5% of the rhGAA is phosphorylatedat the fifth N-glycosylation site (e.g. N596 for SEQ ID NO: 5 and N692for SEQ ID NO: 1). In other embodiments, less than 5%, 10%, 15%, 20% or25% of the rhGAA is phosphorylated at the fifth N-glycosylation site.For example, the fifth N-glycosylation site can have fucosylateddi-antennary complex glycans as the major species. In some embodiments,at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA is sialylated atthe fifth N-glycosylation site.

In one or more embodiments, at least 5% of the rhGAA is phosphorylatedat the sixth N-glycosylation site (e.g. N826 for SEQ ID NO: 5 and N882for SEQ ID NO: 1). In other embodiments, less than 5%, 10%, 15%, 20% or25% of the rhGAA is phosphorylated at the sixth N-glycosylation site.For example, the sixth N-glycosylation site can have a mixture of di-,tri-, and tetra-antennary complex glycans as the major species. In someembodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the rhGAA issialylated at the sixth N-glycosylation site.

In one or more embodiments, at least 5% of the rhGAA is phosphorylatedat the seventh N-glycosylation site (e.g. N869 for SEQ ID NO: 5 and N925for SEQ ID NO: 1). In other embodiments, less than 5%, 10%, 15%, 20% or25% of the rhGAA is phosphorylated at the seventh N-glycosylation site.In some embodiments, less than 40%, 45%, 50%, 55%, 60% or 65% % of therhGAA has any glycan at the seventh N-glycosylation site. In someembodiments, at least 30%, 35% or 40% of the rhGAA has a glycan at theseventh N-glycosylation site.

The recombinant human acid α-glucosidase is preferably produced byChinese hamster ovary (CHO) cells, such as CHO cell line GA-ATB-200 orATB-200-001-X5-14, or by a subculture or derivative of such a CHO cellculture. DNA constructs, which express allelic variants of acidα-glucosidase or other variant acid α-glucosidase amino acid sequencessuch as those that are at least 90%, 95%, 98% or 99% identical to SEQ IDNO: 1 or SEQ ID NO: 5, may be constructed and expressed in CHO cells.These variant acid α-glucosidase amino acid sequences may containdeletions, substitutions and/or insertions relative to SEQ ID NO: 1 orSEQ ID NO: 5, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15 or more deletions, substitutions and/or insertions relative tothe amino acid sequence described by SEQ ID NO: 1 or SEQ ID NO: 5. Thoseof skill in the art can select alternative vectors suitable fortransforming CHO cells for production of such DNA constructs.

Various alignment algorithms and/or programs may be used to calculatethe identity between two sequences, including FASTA, or BLAST which areavailable as a part of the GCG sequence analysis package (University ofWisconsin, Madison, Wis.), and can be used with, e.g., default setting.For example, polypeptides having at least 90%, 95%, 98% or 99% identityto specific polypeptides described herein and preferably exhibitingsubstantially the same functions, as well as polynucleotide encodingsuch polypeptides, are contemplated. Unless otherwise indicated asimilarity score will be based on use of BLOSUM62. When BLASTP is used,the percent similarity is based on the BLASTP positives score and thepercent sequence identity is based on the BLASTP identities score.BLASTP “Identities” shows the number and fraction of total residues inthe high scoring sequence pairs which are identical; and BLASTP“Positives” shows the number and fraction of residues for which thealignment scores have positive values and which are similar to eachother. Amino acid sequences having these degrees of identity orsimilarity or any intermediate degree of identity of similarity to theamino acid sequences disclosed herein are contemplated and encompassedby this disclosure. The polynucleotide sequences of similar polypeptidesare deduced using the genetic code and may be obtained by conventionalmeans, in particular by reverse translating its amino acid sequenceusing the genetic code.

The inventors have found that recombinant human acid α-glucosidasehaving superior ability to target cation-independent mannose-6-phosphatereceptors (CIMPR) and cellular lysosomes as well as glycosylationpatterns that reduce its non-productive clearance in vivo can beproduced using Chinese hamster ovary (CHO) cells. These cells can beinduced to express recombinant human acid α-glucosidase withsignificantly higher levels of N-glycan units bearing one or moremannose-6-phosphate residues than conventional recombinant human acidα-glucosidase products such as alglucosidase alfa. The recombinant humanacid α-glucosidase produced by these cells, for example, as exemplifiedby ATB200, has significantly more muscle cell-targetingmannose-6-phosphate (mono-M6P) and bis-mannose-6-phosphate (bis-M6P)N-glycan residues than conventional acid α-glucosidase, such asLumizyme®. Without being bound by theory, it is believed that thisextensive glycosylation allows the ATB200 enzyme to be taken up moreeffectively into target cells, and therefore to be cleared from thecirculation more efficiently than other recombinant human acidα-glucosidases, such as for example, alglucosidase alfa, which has amuch lower M6P and bis-M6P content. ATB200 has been shown to efficientlybind to CIMPR and be efficiently taken up by skeletal muscle and cardiacmuscle and to have a glycosylation pattern that provides a favorablepharmacokinetic profile and reduces non-productive clearance in vivo.

It is also contemplated that the unique glycosylation of ATB200 cancontribute to a reduction of the immunogenicity of ATB200 compared to,for example, alglucosidase alfa. As will be appreciated by those skilledin the art, glycosylation of proteins with conserved mammalian sugarsgenerally enhances product solubility and diminishes product aggregationand immunogenicity. Glycosylation indirectly alters proteinimmunogenicity by minimizing protein aggregation as well as by shieldingimmunogenic protein epitopes from the immune system (Guidance forIndustry—Immunogenicity Assessment for Therapeutic Protein Products, USDepartment of Health and Human Services, Food and Drug Administration,Center for Drug Evaluation and Research, Center for Biologics Evaluationand Research, August 2014). Therefore, in at least one embodiment,administration of the recombinant human acid α-glucosidase does notinduce anti-drug antibodies. In at least one embodiment, administrationof the recombinant human acid α-glucosidase induces a lower incidence ofanti-drug antibodies in a subject than the level of anti-drug antibodiesinduced by administration of alglucosidase alfa.

As described in co-pending international patent applicationPCT/US2015/053252, cells such as CHO cells can be used to produce therhGAA described therein, and this rhGAA can be used in the presentinvention. Examples of such a CHO cell line are GA-ATB-200 orATB-200-001-X5-14, or a subculture thereof that produces a rhGAAcomposition as described therein. Such CHO cell lines may containmultiple copies of a gene, such as 5, 10, 15, or 20 or more copies, of apolynucleotide encoding GAA.

The high M6P and bis-M6P rhGAA, such as ATB200 rhGAA, can be produced bytransforming CHO cells with a DNA construct that encodes GAA. While CHOcells have been previously used to make rhGAA, it was not appreciatedthat transformed CHO cells could be cultured and selected in a way thatwould produce rhGAA having a high content of M6P and bis-M6P glycanswhich target the CIMPR.

Surprisingly, it was found that it was possible to transform CHO celllines, select transformants that produce rhGAA containing a high contentof glycans bearing M6P or bis-M6P that target the CIMPR, and to stablyexpress this high-M6P rhGAA. Thus, methods for making these CHO celllines are also described in co-pending international patent applicationPCT/US2015/053252. This method involves transforming a CHO cell with DNAencoding GAA or a GAA variant, selecting a CHO cell that stablyintegrates the DNA encoding GAA into its chromosome(s) and that stablyexpresses GAA, and selecting a CHO cell that expresses GAA having a highcontent of glycans bearing M6P or bis-M6P, and, optionally, selecting aCHO cell having N-glycans with high sialic acid content and/or havingN-glycans with a low non-phosphorylated high-mannose content. In atleast one embodiment, the GAA has low levels of complex glycans withterminal galactose.

These CHO cell lines may be used to produce rhGAA and rhGAA compositionsby culturing the CHO cell line and recovering said composition from theculture of CHO cells.

The recombinant human acid α-glucosidase, or a pharmaceuticallyacceptable salt thereof, can be formulated in accordance with theroutine procedures as a pharmaceutical composition adapted foradministration to human beings. For example, in a preferred embodiment,a composition for intravenous administration is a solution in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic to ease pain at thesite of the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampule or sachet indicating the quantity of activeagent. Where the composition is to be administered by infusion, it canbe dispensed with an infusion bottle containing sterile pharmaceuticalgrade water, saline or dextrose/water. Where the composition isadministered by injection, an ampule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

Recombinant human acid α-glucosidase (or a composition or medicamentcontaining recombinant human acid α-glucosidase) is administered by anappropriate route. In one embodiment, the recombinant human acidα-glucosidase is administered intravenously. In other embodiments,recombinant human acid α-glucosidase is administered by directadministration to a target tissue, such as to heart or skeletal muscle(e.g., intramuscular), or nervous system (e.g., direct injection intothe brain; intraventricularly; intrathecally). More than one route canbe used concurrently, if desired.

The recombinant human acid α-glucosidase (or a composition or medicamentcontaining recombinant human acid α-glucosidase) is administered in atherapeutically effective amount (e.g., a dosage amount that, whenadministered at regular intervals, is sufficient to treat the disease,such as by ameliorating symptoms associated with the disease, preventingor delaying the onset of the disease, and/or lessening the severity orfrequency of symptoms of the disease). The amount which will betherapeutically effective in the treatment of the disease will depend onthe nature and extent of the disease's effects, and can be determined bystandard clinical techniques. In addition, in vitro or in vivo assaysmay optionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed will also depend on the route ofadministration, and the seriousness of the disease, and should bedecided according to the judgment of a practitioner and each patient'scircumstances. Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems. In at leastone embodiment, the recombinant human acid α-glucosidase is administeredby intravenous infusion at a dose of about 5 mg/kg to about 30 mg/kg,typically about 5 mg/kg to about 20 mg/kg. In at least one embodiment,the recombinant human acid α-glucosidase is administered by intravenousinfusion at a dose of about 5 mg/kg, about 10 mg/kg, about 15 mg/kg orabout 20 mg/kg. In at least one embodiment, the recombinant human acidα-glucosidase is administered by intravenous infusion at a dose of about20 mg/kg. The effective dose for a particular individual can be varied(e.g., increased or decreased) over time, depending on the needs of theindividual. For example, in times of physical illness or stress, or ifanti-acid α-glucosidase antibodies become present or increase, or ifdisease symptoms worsen, the amount can be increased.

The therapeutically effective amount of recombinant human acidα-glucosidase (or composition or medicament containing recombinant humanacid α-glucosidase) is administered at regular intervals, depending onthe nature and extent of the disease's effects, and on an ongoing basis.Administration at a “regular interval,” as used herein, indicates thatthe therapeutically effective amount is administered periodically (asdistinguished from a one-time dose). The interval can be determined bystandard clinical techniques. In preferred embodiments, recombinanthuman acid α-glucosidase is administered monthly, bimonthly; weekly;twice weekly; or daily. The administration interval for a singleindividual need not be a fixed interval, but can be varied over time,depending on the needs of the individual. For example, in times ofphysical illness or stress, if anti-recombinant human acid α-glucosidaseantibodies become present or increase, or if disease symptoms worsen,the interval between doses can be decreased. In some embodiments, atherapeutically effective amount of 5, 10, 20, 50, 100, or 200 mgenzyme/kg body weight is administered twice a week, weekly or everyother week with or without a chaperone.

The recombinant human acid α-glucosidase of the invention may beprepared for later use, such as in a unit dose vial or syringe, or in abottle or bag for intravenous administration. Kits containing therecombinant human acid α-glucosidase, as well as optional excipients orother active ingredients, such as chaperones or other drugs, may beenclosed in packaging material and accompanied by instructions forreconstitution, dilution or dosing for treating a subject in need oftreatment, such as a patient having Pompe disease.

In at least one embodiment, the miglustat and the recombinant human acidα-glucosidase are administered simultaneously. In at least oneembodiment, the miglustat and the recombinant human acid α-glucosidaseare administered sequentially. In at least one embodiment, the miglustatis administered prior to administration of the recombinant human acidα-glucosidase. In at least one embodiment, the miglustat is administeredless than three hours prior to administration of the recombinant humanacid α-glucosidase. In at least one embodiment, the miglustat isadministered about two hours prior to administration of the recombinanthuman acid α-glucosidase. In at least one embodiment, the miglustat isadministered less than two hours prior to administration of therecombinant human acid α-glucosidase. In at least one embodiment, themiglustat is administered about 1.5 hours prior to administration of therecombinant human acid α-glucosidase. In at least one embodiment, themiglustat is administered about one hour prior to administration of therecombinant human acid α-glucosidase. In at least one embodiment, themiglustat is administered from about 50 minutes to about 70 minutesprior to administration of the recombinant human acid α-glucosidase. Inat least one embodiment, the miglustat is administered from about 55minutes to about 65 minutes prior to administration of the recombinanthuman acid α-glucosidase. In at least one embodiment, the miglustat isadministered about 30 minutes prior to administration of the recombinanthuman acid α-glucosidase. In at least one embodiment, the miglustat isadministered from about 25 minutes to about 35 minutes prior toadministration of the recombinant human acid α-glucosidase. In at leastone embodiment, the miglustat is administered from about 27 minutes toabout 33 minutes prior to administration of the recombinant human acidα-glucosidase.

In at least one embodiment, the miglustat is administered concurrentlywith administration of the recombinant human acid α-glucosidase. In atleast one embodiment, the miglustat is administered within 20 minutesbefore or after administration of the recombinant human acidα-glucosidase. In at least one embodiment, the miglustat is administeredwithin 15 minutes before or after administration of the recombinanthuman acid α-glucosidase. In at least one embodiment, the miglustat isadministered within 10 minutes before or after administration of therecombinant human acid α-glucosidase. In at least one embodiment, themiglustat is administered within 5 minutes before or afteradministration of the recombinant human acid α-glucosidase.

In at least one embodiment, the miglustat is administered afteradministration of the recombinant human acid α-glucosidase. In at leastone embodiment, the miglustat is administered up to 2 hours afteradministration of the recombinant human acid α-glucosidase. In at leastone embodiment, the miglustat is administered about 30 minutes afteradministration of the recombinant human acid α-glucosidase. In at leastone embodiment, the miglustat is administered about one hour afteradministration of the recombinant human acid α-glucosidase. In at leastone embodiment, the miglustat is administered about 1.5 hours afteradministration of the recombinant human acid α-glucosidase. In at leastone embodiment, the miglustat is administered about 2 hours afteradministration of the recombinant human acid α-glucosidase.

Another aspect of the invention provides a kit for combination therapyof Pompe disease in a patient in need thereof. The kit includes apharmaceutically acceptable dosage form comprising miglustat, apharmaceutically acceptable dosage form comprising a recombinant humanacid α-glucosidase as defined herein, and instructions for administeringthe pharmaceutically acceptable dosage form comprising miglustat and thepharmaceutically acceptable dosage form comprising the recombinant acidα-glucosidase to a patient in need thereof. In at least one embodiment,the pharmaceutically acceptable dosage form comprising miglustat is anoral dosage form as described herein, including but not limited to atablet or a capsule. In at least one embodiment, the pharmaceuticallyacceptable dosage form comprising a recombinant human acid α-glucosidaseis a sterile solution suitable for injection as described herein. In atleast one embodiment, the instructions for administering the dosageforms include instructions to administer the pharmaceutically acceptabledosage form comprising miglustat orally prior to administering thepharmaceutically acceptable dosage form comprising the recombinant humanacid α-glucosidase by intravenous infusion, as described herein.

Without being bound by theory, it is believed that miglustat acts as apharmacological chaperone for the recombinant human acid α-glucosidaseATB200 and binds to its active site. Thus, as seen in FIG. 1, miglustathas been found to decrease the percentage of unfolded ATB200 protein andstabilize the active conformation of ATB200, preventing denaturation andirreversible inactivation at the neutral pH of plasma and allowing it tosurvive conditions in the circulation long enough to reach and be takenup by tissues. However, the binding of miglustat to the active site ofATB200 also can result in inhibition of the enzymatic activity of ATB200by preventing the natural substrate, glycogen, from accessing the activesite. It is believed that when miglustat and the recombinant human acidα-glucosidase are administered to a patient under the conditionsdescribed herein, the concentrations of miglustat and ATB200 within theplasma and tissues are such that ATB200 is stabilized until it can betaken up into the tissues and targeted to lysosomes, but, because of therapid clearance of miglustat, hydrolysis of glycogen by ATB200 withinlysosomes is not overly inhibited by the presence of miglustat, and theenzyme retains sufficient activity to be therapeutically useful.

All the embodiments described above may be combined. This includes inparticular embodiments relating to:

the nature of the pharmacological chaperone, for example miglustat; andthe active site for which it is specific;

the dosage, route of administration of the pharmacological chaperone(miglustat) and the type of pharmaceutical composition including thenature of the carrier and the use of commercially availablecompositions;

the nature of the drug, e.g. therapeutic protein drug product, which maybe a counterpart of an endogenous protein for which expression isreduced or absent in the subject, suitably recombinant human acidα-glucosidase, for example the recombinant human acid α-glucosidaseexpressed in Chinese hamster ovary (CHO) cells and comprising anincreased content of N-glycan units bearing one or moremannose-6-phosphate residues when compared to a content of N-glycanunits bearing one or more mannose-6-phosphate residues of alglucosidasealfa; and suitably having an amino acid sequence as set forth in SEQ IDNO: 1, SEQ ID NO: 2 (or as encoded by SEQ ID NO: 2), SEQ ID NO: 3, SEQID NO: 4 or SEQ ID NO: 5;

the number and type of N-glycan units on the recombinant human acidα-glucosidase, e.g. N-acetylglucosamine, galactose, sialic acid orcomplex N-glycans formed from combinations of these) attached to therecombinant human acid α-glucosidase;

the degree of phosphorylation of mannose units on the recombinant humanacid α-glucosidase to form mannose-6-phosphate and/orbis-mannose-6-phosphate;

the dosage and route of administration (e.g. intravenous administration,especially intravenous infusion, or direct administration to the targettissue) of the replacement enzyme (recombinant human acid α-glucosidase)and the type of formulation including carriers and therapeuticallyeffective amount;

the dosage interval of the pharmacological chaperone (miglustat) and therecombinant human acid α-glucosidase;

the nature of the therapeutic response and the results of thecombination therapy (e.g. enhanced results as compared to the effect ofeach therapy performed individually);

the timing of the administration of the combination therapy, e.g.simultaneous administration of miglustat and the recombinant human acidα-glucosidase or sequential administration, for example wherein themiglustat is administered prior to the recombinant human acidα-glucosidase or after the recombinant human acid α-glucosidase orwithin a certain time before or after administration of the recombinanthuman acid α-glucosidase; and

the nature of the patient treated (e.g. mammal such as human) and thecondition suffered by the individual (e.g. enzyme insufficiency).

Any of the embodiments in the list above may be combined with one ormore of the other embodiments in the list.

EXAMPLES

Other features of the present invention will become apparent from thefollowing non-limiting examples which illustrate, by way of example, theprinciples of the invention.

Example 1 Limitations of Existing Myozyme® and Lumizyme® rhGAA Products

To evaluate the ability of the rhGAA in Myozyme® and Lumizyme®, the onlycurrently approved treatments for Pompe disease, these rhGAApreparations were injected onto a CIMPR column (which binds rhGAA havingM6P groups) and subsequently eluted with a free M6 gradient. Fractionswere collected in 96-well plate and GAA activity assayed by4MU-α-glucose substrate. The relative amounts of bound and unbound rhGAAwere determined based on GAA activity and reported as the fraction oftotal enzyme.

FIGS. 2A-B describe the problems associated with conventional ERTs(Myozyme® and Lumizyme®): 73% of the rhGAA in Myozyme® (FIG. 2B) and 78%of the rhGAA in Lumizyme® (FIG. 2A) did not bind to the CIMPR, see theleft-most peaks in each figure. Only 27% of the rhGAA in Myozyme® and22% of the rhGAA in Lumizyme® contained M6P that can productive totarget it to the CIMPR on muscle cells.

An effective dose of Myozyme® and Lumizyme® corresponds to the amount ofrhGAA containing M6P which targets the CIMPR on muscle cells. However,most of the rhGAA in these two conventional products does not target theCIMPR receptor on target muscle cells. The administration of aconventional rhGAA where most of the rhGAA is not targeted to musclecells increases the risk of allergic reaction or induction of immunityto the non-targeted rhGAA.

Example 2 Preparation of CHO Cells Producing ATB200 rhGAA Having a HighContent of Mono- or Bis-M6P-Bearing N-Glycans

CHO cells were transfected with DNA that expresses rhGAA followed byselection of transformants producing rhGAA. A DNA construct fortransforming CHO cells with DNA encoding rhGAA is shown in FIG. 3. CHOcells were transfected with DNA that expresses rhGAA followed byselection of transformants producing rhGAA.

After transfection, DG44 CHO (DHFR−) cells containing a stablyintegrated GAA gene were selected with hypoxanthine/thymidine deficient(−HT) medium). Amplification of GAA expression in these cells wasinduced by methotrexate treatment (MTX, 500 nM). Cell pools thatexpressed high amounts of GAA were identified by GAA enzyme activityassays and were used to establish individual clones producing rhGAA.Individual clones were generated on semisolid media plates, picked byClonePix system, and were transferred to 24-deep well plates. Theindividual clones were assayed for GAA enzyme activity to identifyclones expressing a high level of GAA. Conditioned media for determiningGAA activity used a 4-MU-α-glucopyranoside α-glucosidase substrate.Clones producing higher levels of GAA as measured by GAA enzyme assayswere further evaluated for viability, ability to grow, GAA productivity,N-glycan structure and stable protein expression. CHO cell lines,including CHO cell line GA-ATB-200, expressing rhGAA with enhancedmono-M6P or bis-M6P N-glycans were isolated using this procedure.

Example 3 Capturing and Purification of ATB200 rhGAA

Multiple batches of the rhGAA according to the invention were producedin shake flasks and in perfusion bioreactors using CHO cell lineGA-ATB-200 and CIMPR binding was measured. Similar CIMPR receptorbinding (˜70%) to that shown in FIG. 4B and FIG. 5A was observed forpurified ATB200 rhGAA from different production batches indicating thatATB200 rhGAA can be consistently produced. As shown by FIGS. 2A, 2B, 4Aand 4B, Myozyme® and Lumizyme® rhGAAs exhibited significantly less CIMPRbinding than ATB200 rhGAA.

Example 4 Analytical Comparison of ATB200 to Lumizyme®

Weak anion exchange (“WAX”) liquid chromatography was used tofractionate ATB200 rhGAA according to terminal phosphate. Elutionprofiles were generated by eluting the ERT with increasing amount ofsalt. The profiles were monitored by UV (A280 nm). ATB200 rhGAA wasobtained from CHO cells and purified. Lumizyme® was obtained from acommercial source. Lumizyme® exhibited a high peak on the left of itselution profile. ATB200 rhGAA exhibited four prominent peaks eluting tothe right of Lumizyme® (FIG. 6). This confirms that ATB200 rhGAA wasphosphorylated to a greater extent than Lumizyme® since this evaluationis by terminal charge rather than CIMPR affinity.

Example 5 Oligosaccharide Characterization of ATB200 rhGAA

Purified ATB200 rhGAA and Lumizyme® glycans were evaluated by MALDI-TOFto determine the individual glycan structures found on each ERT (FIG.7). ATB200 samples were found to contain lower amounts ofnon-phosphorylated high-mannose type N-glycans than Lumizyme®. Thehigher content of M6P glycans in ATB200 than in Lumizyme®, targetsATB200 rhGAA to muscle cells more effectively. The high percentage ofmono-phosphorylated and bis-phosphorylated structures determined byMALDI agree with the CIMPR profiles which illustrated significantlygreater binding of ATB200 to the CIMPR receptor. N-glycan analysis viaMALDI-TOF mass spectrometry confirmed that on average each ATB200molecule contains at least one natural bis-M6P N-glycan structure. Thishigher bis-M6P N-glycan content on ATB200 rhGAA directly correlated withhigh-affinity binding to CIMPR in M6P receptor plate binding assays (KDabout 2-4 nM) FIG. 9A.

ATB200 rhGAA was also analyzed for site-specific N-glycan profiles usingtwo different LC-MS/MS analytical techniques. In the first analysis, theprotein was denatured, reduced, alkylated and digested prior to LC-MS/MSanalysis. During protein denaturation and reduction, 200 μg of proteinsample, 5 μL 1 mol/L tris-HCl (final concentration 50 mM), 75 μL 8 mol/Lguanidine HCl (final concentration 6 M), 1 μL 0.5 mol/L EDTA (finalconcentration 5 mM), 2 μL 1 mol/L DTT (final concentration 20 mM) andMilli-Q® water were added to a 1.5 mL tube to provide a total volume of100 μL. The sample was mixed and incubated at 56° C. for 30 minutes in adry bath. During alkylation, the denatured and reduced protein samplewas mixed with 5 μL 1 mol/L iodoacetamide (IAM, final concentration 50mM), then incubated at 10-30° C. in the dark for 30 minutes. Afteralkylation, 400 μL of precooled acetone was added to the sample and themixture was frozen at −80° C. refrigeration for 4 hours. The sample wasthen centrifuged for 5 min at 13000 rpm at 4° C. and the supernatant wasremoved. 400 μL of precooled acetone was added to the pellets, which wasthen centrifuged for 5 min at 13000 rpm at 4° C. and the supernatant wasremoved. The sample was then air dried on ice in the dark to removeacetone residue. 40 μL of 8M urea and 160 μL of 100 mM NH₄HCO₃ wereadded to the sample to dissolve the protein. During trypsin digestion,50 μg of the protein was then added with trypsin digestion buffer to afinal volume of 100 μL, and 5 μL 0.5 mg/mL trypsin (protein to enzymeratio of 20/1 w/w) was added. The solution was mixed well and incubatedovernight (16±2 hours) at 37° C. 2.5 μL 20% TFA (final concentration0.5%) was added to quench the reaction. The sample was then analyzedusing the Thermo Scientific Orbitrap Velos Pro™ Mass Spectrometer.

In the second LC-MS/MS analysis, the ATB200 sample was preparedaccording to a similar denaturation, reduction, alkylation and digestionprocedure, except that iodoacetic acid (IAA) was used as the alkylationreagent instead of IAM, and then analyzed using the Thermo ScientificOrbitrap Fusion Lumos Tribid™ Mass Spectrometer.

In a third LC-MS/MS analysis, the ATB200 sample was prepared accordingto a similar denaturation, reduction, alkylation and digestion procedureusing iodoacetamide (IAM) as the alkylation reagent, and then analyzedusing the Thermo Scientific Orbitrap Fusion Mass Spectrometer.

The results of the first and second analyses are shown in FIGS. 8B-8Hand the result of the third analysis is shown in FIG. 8A. In FIGS.8B-8H, the results of the first analysis are represented by left bar(dark grey) and the results from the second analysis are represented bythe right bar (light grey). In FIGS. 8B-8H, the symbol nomenclature forglycan representation is in accordance with Varki, A., Cummings, R. D.,Esko J. D., et al., Essentials of Glycobiology, 2nd edition (2009). InFIGS. 8A-8H, the glycosylation sites are given relative to SEQ ID NO: 5:N84, N177, N334, N414, N596, N826 and N869. For the full-length aminoacid sequence of SEQ ID NO: 1, these potential glycosylation sites areat the following positions: N140, N233, N390, N470, N652, N882 and N925.

As can be seen from FIGS. 8B-8H, the first two analyses provided similarresults, although there was some variation between the results. Thisvariation can be due to a number of factors, including the instrumentused and the completeness of N-glycan analysis. For example, if somespecies of phosphorylated glycans were not identified and/or notquantified, then the total number of phosphorylated glycans may beunderrepresented, and the percentage of rhGAA bearing the phosphorylatedglycans at that site may be underrepresented. As another example, ifsome species of non-phosphorylated glycans were not identified and/ornot quantified, then the total number of non-phosphorylated glycans maybe underrepresented, and the percentage of rhGAA bearing thephosphorylated glycans at that site may be overrepresented.

FIG. 8A shows the N-glycosylation site occupancy of ATB200. As can beseen from FIG. 8A, the first, second, third, fourth, fifth and sixthN-glycosylation sites are mostly occupied, with approximately 90% and upto about 100% of the ATB200 enzyme having a glycan detected at eachpotential site. However, the seventh potential N-glycosylation site isglycosylated about half of the time.

FIG. 8B shows the N-glycosylation profile of the first site, N84. As canbe seen from FIG. 8B, the major glycan species is bis-M6P glycans. Boththe first and second analyses detected over 75% of the ATB200 had abis-M6P glycan at the first site.

FIG. 8C shows the N-glycosylation profile of the second site, N177. Ascan be seen from FIG. 8C, the major glycan species are mono-M6P glycansand non-phosphorylated high mannose glycans. Both the first and secondanalyses detected over 40% of the ATB200 had a mono-M6P glycan at thesecond site.

FIG. 8D shows the N-glycosylation profile of the third site, N334. Ascan be seen from FIG. 8D, the major glycan species arenon-phosphorylated high mannose glycans, di-, tri-, and tetra-antennarycomplex glycans, and hybrid glycans. Both the first and second analysesdetected over 20% of the ATB200 had a sialic acid residue at the thirdsite.

FIG. 8E shows the N-glycosylation profile of the fourth site, N414. Ascan be seen from FIG. 8E, the major glycan species are bis-M6P andmono-M6P glycans. Both the first and second analyses detected over 40%of the ATB200 had a bis-M6P glycan at the fourth site. Both the firstand second analyses also detected over 25% of the ATB200 had a mono-M6Pglycan at the fourth site.

FIG. 8F shows the N-glycosylation profile of the fifth site, N596. Ascan be seen from FIG. 8F, the major glycan species are fucosylateddi-antennary complex glycans. Both the first and second analysesdetected over 70% of the ATB200 had a sialic acid residue at the fifthsite.

FIG. 8G shows the N-glycosylation profile of the sixth site, N826. Ascan be seen from FIG. 8G, the major glycan species are di-, tri-, andtetra-antennary complex glycans. Both the first and second analysesdetected over 80% of the ATB200 had a sialic acid residue at the sixthsite.

FIG. 8H shows a summary of the phosphorylation at each of the first sixpotential N-glycosylation sites. As can be seen from FIG. 8H, both thefirst and second analyses detected high phosphorylation levels at thefirst, second and fourth sites. Both analyses detected over 80% of theATB200 was mono- or di-phosphorylated at the first site, over 40% of theATB200 was mono-phosphorylated at the second site, and over 80% of theATB200 was mono- or di-phosphorylated at the fourth site.

Example 6 Characterization of CIMPR Affinity of ATB200

In addition to having a greater percentage of rhGAA that can bind to theCIMPR, it is important to understand the quality of that interaction.Lumizyme® and ATB200 rhGAA receptor binding was determined using a CIMPRplate binding assay. Briefly, CIMPR-coated plates were used to captureGAA. Varying concentrations of rhGAA were applied to the immobilizedreceptor and unbound rhGAA was washed off. The amount of remaining rhGAAwas determined by GAA activity. As shown by FIG. 9A, ATB200 rhGAA boundto CIMPR significantly better than Lumizyme®.

FIG. 9B shows the relative content of bis-M6P glycans in Lumizyme®, aconventional rhGAA, and ATB200 according to the invention. For Lumizyme®there is on average only 10% of molecules have a bis-phosphorylatedglycan. Contrast this with ATB200 where on average every rhGAA moleculehas at least one bis-phosphorylated glycan.

Example 7 ATB200 rhGAA was More Efficiently Internalized by Fibroblastthan Lumizyme®

The relative cellular uptake of ATB200 and Lumizyme® rhGAA were comparedusing normal and Pompe fibroblast cell lines. Comparisons involved 5-100nM of ATB200 rhGAA according to the invention with 10-500 nMconventional rhGAA Lumizyme®. After 16-hr incubation, external rhGAA wasinactivated with TRIS base and cells were washed 3-times with PBS priorto harvest. Internalized GAA measured by 4MU-α-Glucoside hydrolysis andwas graphed relative to total cellular protein and the results appear inFIGS. 10A-B.

ATB200 rhGAA was also shown to be efficiently internalized into cells(FIGS. 10A and 10B), respectively, show that ATB200 rhGAA isinternalized into both normal and Pompe fibroblast cells and that it isinternalized to a greater degree than conventional Lumizyme® rhGAA.ATB200 rhGAA saturates cellular receptors at about 20 nM, while about250 nM of Lumizyme® is needed. The uptake efficiency constant (Kuptake)extrapolated from these results is 2-3 nm for ATB200 and 56 nM forLumizyme® as shown by FIG. 10C. These results suggest that ATB200 rhGAAis a well-targeted treatment for Pompe disease.

Example 8 Population Pharmacokinetic (PK) Modeling for ATB200 andMiglustat

Pharmacokinetic data for acid α-glucosidase (ATB200), including samplingtimes, dosing history and plasma concentrations of acid α-glucosidase,is obtained from mice, rats and monkeys administered ATB200 byintravenous injection. Pharmacokinetic data for miglustat andduvoglustat in plasma and tissue is collected from humans or from mice.Modeling and simulations are performed using Phoenix® NLME™ v1.3.Compartmental PK models are constructed to assess the PK of ATB200 inplasma. The models include:

-   -   Description of the relationships between plasma concentration        and time;    -   A variance component characterizing between- and within-animal        variability in model parameters; and    -   A component describing uncertainty in the state of knowledge        about critical model components.

Non-linear mixed effects (NLME) models have the form:

C _(p) _(ij) =C(D _(i) ,t _(j),θ_(i))+ε_(ij)

θ_(i)=(θ_(i1), . . . , θ_(im))

where C_(pij) is concentration at j^(th) collection time (t_(j)) foranimal i, D_(i) represents the dosing history for animal i, θ_(i) is thevector of PK parameters for animal i, and ε_(ij) is the random errorassociated with j^(th) concentration for animal i.

Between-subject variability (BSV) in parameters are modeled as alog-normal distribution:

θ_(in)=θ_(TVn) exp(η_(in))

(η₁, . . . , η_(m))˜MVN(0,Ω)

where θ_(TVn) is the population typical value for the n^(th) PKparameter (e.g. clearance) and η_(in) is the random inter-animal effecton the n^(th) parameter for animal i. Random effects (η₁, . . . , η_(m))were normally distributed with mean 0 and estimated variance ω² includedin the OMEGA (Ω) matrix.

PK is assumed to be species independent and is scaled according to ageneralized Dedrick approach that scales the disposition according tothe power of an animal's body weight:

CL_((p)i)=a_((p))BW_(i) ^(b)

V_((p)i)=c_((p))BW_(i) ^(d)

where CL=systemic clearance, V=volume of distribution, BW=body weight,p=peripheral, b and d=allometric exponents, and a and c=typical valuesfor a BW=1. In this scenario, the exponent b and d can be compared tomore generalized values accepted in the literature (b=0.75 and d=1.0).Nominal BW (0.025, 0.25 and 2.5 kg) are used in the analyses.

Baseline acid α-glucosidase concentration is modeled asC_(baseline)=Rate of acid α-glucosidase synthesis/CL and can beextrapolated to humans, since C_(baseline) is species specific,independent of the concentration of ATB200 and known in humans withPompe disease. A base model is determined using Phoenix® FOCE-ELS, toevaluate whether a 1 or 2 compartment model is best to fit the data.Sources of variability in PK of acid α-glucosidase are also exploredvisually and by searching the effect of the various wildtype/species/dose related effects on PK.

For ATB200, a two-compartment model with linear elimination adequatelycharacterizes the concentration-time profiles of acid α-glucosidaseactivity for all dose levels across animal species. The model includes atheoretical allometric component accounting for difference in bodyweight across animal species on clearance (CL) and volume ofdistribution (Vc). The goodness of fit of the population PK model forATB200 is shown in FIG. 11. Population PK parameters of ATB200 innonclinical studies are presented in Table 1.

TABLE 1 Typical Values Between-subject PK Parameter (Relative standarderror (%)) variability (%) Systemic clearance 0.00957 × (BW/0.25)^(0.78)21.0 (CL; L/h) (5.1) (3.2) Central volume of 0.0101 × (BW/0.25)^(0.83) 5.3 distribution (4.3) (1.7) (V_(c); L) Peripheral clearance 0.000290 ×(BW/0.25)^(0.78) NA (CL_(d); L/h) (43.2) Peripheral volume of 0.000653 ×(BW/0.25)^(0.83) NA distribution (V2; L) (35.6) Endogenous Mouse:0.00401 (8.1) NA rate of acid Rat: 0.0203 (13.3) α-glucosidase Monkey:0.00518 (16.9) synthesis (SYNT; mg/h) BW: body weight

Concentration-time profiles of miglustat (200 mg) in Pompe diseasepatients are compared to those obtained following administration ofduvoglustat in normal healthy volunteers (dose range: 50, 100, 250, 600,and 1000 mg). Dose-normalized plasma concentration-time profiles ofmiglustat and duvoglustat are shown in FIG. 12. Becauseconcentration-time profiles of miglustat in patients with Pompe diseaseare similar to those observed following dosing of duvoglustat in healthysubjects over 24 h, PK data collected for duvoglustat in peripheraltissues were used as a surrogate to model exposure to miglustat. Atwo-compartment model with linear elimination is used to characterizethe concentration-time profiles of duvoglustat in tissues.

Goodness of fit of the PK model of duvoglustat is shown in FIGS. 13A and13B. Final model PK parameters of duvoglustat in plasma and tissue areshown in Table 2.

TABLE 2 Typical Values PK Parameter (CV %) Volume of distribution (V; L)44.5 (7.41) Systemic clearance (CL; L/h) 9.44 (6.99) Rate constant ofabsorption (K_(a); 1/h)) 1.10 (14.0) Peripheral volume of distribution(V2; L) 8.68 (19.39) Central compartment clearance (CL2; L/h) 0.205(23.7) Intercompartment volume of distribution (VQ; L) 61.8 (21.2)Elimination rate constant (Keo) 0.378 (11.1) Intercompartment volume ofdistribution within 3390 central compartment (VQ2; L) Peripheralcompartment clearance (CL3; L/h) 88.0 (7.72) Apparent intercompartmentclearance (CLQ; L/h) 40.6 (10.6) Lag time (h) 0.176 (30.7) Relativestandard error of central compartment 0.477 (6.56) Relative standarderror of peripheral compartment 0.368 (8.19) CV: coefficient ofvariability

A population PK model of miglustat is constructed based on oral dosingin Gaa knockout (KO) mice. Population PK parameters of miglustat in GaaKO mice are presented in Table 3. Goodness of fit is shown in FIG. 14.The model has a residual additive error of 0.475 ng/mL.

TABLE 3 PK Parameter Typical Values (BSV %) Rate constant of absorption(K_(a); h⁻¹) 2.09 (4.56) Systemic clearance (CL; mL/h) 43.3 (9.61)Central volume of distribution (V_(c); mL) 4.55 (45.1) Peripheralclearance (CL_(d); mL/h) 4.57 (32.1) Peripheral volume of distribution(V2; ml) 19.6 (23.3) BSV: between-subject variability

Example 9 Modeling of Recombinant Acid α-Glucosidase (ATB200)Pharmacokinetic (PK) Parameters in Humans

Pharmacokinetic models (Example 8) were used to perform simulations andto predict concentration-time profiles of acid α-glucosidase in humansubjects with late stage Pompe disease following dosing of ATB200. Theallometric function allowed the linkage of body weight to clearance andvolume of distribution, and therefore allowed the prediction of PKparameters in a typical human subjects with a body weight of 70 kg. Themodel is customized by including an endogenous rate of synthesis of acidα-glucosidase in humans (Umapathysivam K, Hopwood J J, Meikle P J.Determination of acid alpha-glucosidase activity in blood spots as adiagnostic test for Pompe disease. Clin Chem. (2001) August; 47(8):1378-83).

A single 20 mg/kg IV dose of ATB200 in humans over a 4-h infusion ispredicted to result in the concentration-time profile presented in FIG.15. PK parameters in a typical 70-kg human and the resulting exposureparameters following a 20 mg/kg IV infusion of ATB200 over 4 h arepresented in Table 4.

TABLE 4 Pharmacokinetic parameter Predicted value Systemic clearance(CL; L/h) 0.768 Central volume of distribution (V_(c); L) 1.09 Areaunder the curve, extrapolated to infinity 1822 (AUC_(0-inf); mg · h/L)Maximum concentration (C_(max); mg/L) 423 Time at which maximumconcentration is achieved 4 (T_(max); h) Half-life (T_(1/2); h) 2.17

The predicted systemic clearance (CL) and volume of distribution (V) ofATB200 in a typical 70-kg patient are 0.768 L/h and 2.41 L,respectively.

According to the product label for Lumizyme® (alglucosidase alfa), thesystemic clearance of acid α-glucosidase at Week 52 following repeateddosing of Lumizyme® in patients with late-stage Pompe disease is 601mL/h (0.601 L/h) and the half-life of Lumizyme® is 2.4 h. Based on theabove model, the systemic clearance of ATB200 in adult subjects withPompe disease is expected to be approximately 28% faster than thatreported for Lumizyme®. In addition, the predicted AUC in humansfollowing a 20 mg/kg dose of ATB200 is expected to be about 25% lower(AUC_(0-inf): 1822 mg·h/L) than the AUC reported following a 20 mg/kgdose of Lumizyme® (2700 μg·h/mL).

Example 10 Exposure-Response Models for Glycogen Reduction

Gaa knockout mice are administered acid α-glucosidase (ATB200)intravenously at doses of 5, 10 and 20 mg/kg, rising oral doses ofmiglustat (1, 3 and 10 mg/kg) concomitantly with intravenous doses of 5or 10 mg/kg of ATB200 or rising oral doses of miglustat (1, 3, 5, 10,20, and 30 mg/kg) concomitantly with intravenous doses of 20 mg/kg ofATB200. Glycogen levels are measured as previously described (Khanna, R,Flanagan, J J, Feng, J, Soska, R, Frascella, M, Pellegrino, L J et al.(2012). “The pharmacological chaperone AT2220 increases recombinanthuman acid α-glucosidase uptake and glycogen reduction in a mouse modelof Pompe disease. PLoS One 7(7): e40776). The ratios of glycogen levelsobserved after each combination therapy treatment to the glycogen levelobserved after monotherapy (glycogen ratio) are calculated. Results areprovided in Table 5.

TABLE 5 Glycogen (μg/mg protein) Combination Therapy Monotherapy StudyStudy Treatments Study #1 #2 #3 ATB200 Miglustat Median Median MedianMedian Ratio (mg/kg) (mg/kg) (N) (N) (N) (N) (Combination/Monotherapy) 5NA 307 NA NA 307 NA (N = 7) (N = 7) 10 NA 259 NA NA 259 NA (N = 7) (N =7) 20 NA 157 NA 195 181 NA (N = 7) (N = 14) (N = 21) 5 1 NA 323 NA 3231.05 (N = 7) (N = 7) 3 NA 359 NA 359 1.17 (N = 6) (N = 6) 10 NA 352 NA352 1.15 (N = 7) (N = 7) 10 1 NA 273 NA 273 1.05 (N = 7) (N = 7) 3 NA252 NA 252 0.973 (N = 7) (N = 7) 10 NA 278 NA 278 1.07 (N = 7) (N = 7)20 1 NA 154 NA 154 0.851 (N = 7) (N = 7) 3 NA 175 NA 175 0.967 (N = 7)(N = 7) 5 NA NA 163 163 0.900 (N = 14) (N = 14) 10 NA  97 145 118 0.652(N = 6) (N = 13) (N = 19) 20 NA NA 122 122 0.674 (N = 13) (N = 13) 30 NA167 175 170 0.939 (N = 6) (N = 14) (N = 20)

In addition, FIGS. 15A to 15C show the effects of administeringalglucosidase alfa (Lumizyme®) and ATB200 on glycogen clearance in Gaaknockout mice. Animals are given two IV bolus administrations (everyother week); tissues are harvested two weeks after the last dose andanalyzed for acid α-glucosidase activity and glycogen content.

As seen from the results in Table 5, ATB200 was found to deplete tissueglycogen in acid α-glucosidase (Gaa) knockout mice in a dose-dependentfashion. The 20 mg/kg dose of ATB200 consistently removed a greaterproportion of stored glycogen in Gaa knockout mice than the 5 and 10mg/kg dose levels. However, as seen in FIGS. 15A to 15C, ATB200administered at 5 mg/kg showed a similar reduction of glycogen in mouseheart and skeletal muscles (quadriceps and triceps) to Lumizyme®administered at 20 mg/kg, while ATB200 dosed at 10 and 20 mg/kg showedsignificantly better reduction of glycogen levels in skeletal musclesthan Lumizyme®.

Furthermore, 10 and 20 mg/kg doses of miglustat co-administered withATB200 at 20 mg/kg resulted in reduction of glycogen levels in Gaaknockout mice to 118 and 122 μg/mg protein, respectively. Dosing ofmiglustat at 30 mg/kg caused less reduction of glycogen. Without beingbound by theory, it is believed that at higher concentrations ofmiglustat, inhibition of acid α-glucosidase in lysosomes may exceed thebeneficial chaperone effect, thus reducing degradation of glycogen inthe lysosome.

Pharmacokinetic models (Example 8) are used to predict exposure to acidα-glucosidase and miglustat, time-matched to the values for tissuelysosomal glycogen levels in Table 5. Steady state exposure (AUC) ratios(average exposure over 24 hours) of miglustat/ATB200 are derived foreach treatment combination tested, plotted against the correspondingglycogen ratio (Table 5) and fitted to a mathematical function. Theexposure-response curve is shown in FIG. 17.

As seen from the results in FIG. 17, co-administration of 10 and 20mg/kg doses of miglustat with a 20 mg/kg dose of ATB200 provides goodstability of acid α-glucosidase activity in plasma, while maximizingglycogen reduction. Lower doses of miglustat (1, 3, and 5 mg/kg) arebelieved to result in sub-optimal stabilization of acid α-glucosidaseactivity, whereas the highest dose of miglustat (30 mg/kg) is believedto result in excessive inhibition of α-glucosidase activity withinlysosomes.

Based on pharmacokinetic models (Example 8), the observedmiglustat/ATB200 AUC ratio of 0.01159 (10 mg/kg miglustatco-administered with 20 mg/kg ATB200) is expected to correspond to amiglustat dose of about 270 mg co-administered with 20 mg/kg ATB200 in atypical 70-kg human. AUC ratios of 0.01 and 0.02 would correspond tomiglustat doses of 233 and 466 mg, respectively, co-administered with 20mg/kg ATB200, in a typical 70-kg subject.

Example 11 Modeling of Miglustat/Duvoglustat Concentrations in Humans

Pharmacokinetic models (Example 8) were used to predict the length oftime that the plasma or tissue concentrations of duvoglustat (asurrogate of miglustat) would remain above the IC₅₀ (the concentrationgiving 50% of maximum inhibition of acid α-glucosidase activity) ofmiglustat in plasma and lysosome. Inhibition of acid α-glucosidaseactivity is determined by methods described previously (Flanagan J J,Rossi B, Tang K, Wu X, Mascioli K, et al. (2009) “The pharmacologicalchaperone 1-deoxynojirimycin increases the activity and lysosomaltrafficking of multiple mutant forms of acid alpha-glucosidase.” HumMutat 30: 1683-1692). The IC₅₀ value of miglustat at the pH of plasma(pH 7.0) was determined to be 170 μg/L, while the IC₅₀ value at the pHof the lysosomal compartment (pH 5.2) was determined to be 377 μg/L.

Results of the model prediction are presented in Table 6. Predictedconcentration-time profiles of miglustat in plasma and lysosomesfollowing repeated dosing are shown in FIGS. 17 and 18, respectively.

TABLE 6 Miglustat Dose Time > IC₅₀ (h) (mg) Plasma (pH 7.0) Lysosome (pH5.2) 100 13.1 0 150 15.0 0 200 16.4 1.19 233 17.2 2.96 250 17.5 3.58 27017.9 4.15 300 18.4 4.92 466 20.7 8.04 600 22.0 9.96 699 22.8 11.2

Based on the results presented in Table 6 and FIGS. 17 and 18, a 260 mgdose of miglustat is expected to bind to and stabilize ATB200 in plasmaup to 18 hours whereas inhibition of acid α-glucosidase activity in thelysosome is expected to last only 4 hours.

Example 12 Muscle Physiology and Morphology in Gaa-Knockout Mice

Gaa knockout mice are given two IV bolus administrations of recombinanthuman acid α-glucosidase (alglucosidase alfa or ATB200) at 20 mg/kgevery other week. Miglustat is orally administered at dosages of 10mg/kg to a subset of animals treated with ATB200 30 mins prior toadministration of ATB200. Control mice are treated with vehicle alone.Soleus, quadriceps and diaphragm tissue is harvested two weeks after thelast dose of recombinant human acid α-glucosidase. Soleus and diaphragmtissue are analyzed for glycogen levels, by staining with periodicacid-Schiff reagent (PAS), and for lysosome proliferation, by measuringlevels of the lysosome-associated membrane protein (LAMP1) marker, whichis upregulated in Pompe disease. Semi-thin sections of quadriceps muscleembedded in epoxy resin (Epon) are stained with methylene blue andobserved by electron microscopy (1000×) to determine the extent of thepresence of vacuoles. Quadriceps muscle samples are analyzedimmunohistochemically to determine levels of the autophagy markersmicrotubule-associated protein 1A/1B-light chain 3phosphatidylethanolamine conjugate (LC3A II) and p62, theinsulin-dependent glucose transporter GLUT4 and the insulin-independentglucose transporter GLUT1.

In a similar study, Gaa knockout mice are given four IV bolusadministrations of recombinant human acid α-glucosidase (alglucosidasealfa or ATB200) at 20 mg/kg every other week. Miglustat is orallyadministered at dosages of 10 mg/kg to a subset of animals treated withATB200 30 mins prior to administration of ATB200. Control mice aretreated with vehicle alone. Cardiac muscle tissue is harvested two weeksafter the last dose of recombinant human acid α-glucosidase and analyzedfor glycogen levels, by staining with periodic acid-Schiff reagent(PAS), and for lysosome proliferation, by measuring levels of LAMP1.

As seen in FIG. 20, administration of ATB200 showed a reduction inlysosome proliferation in heart, diaphragm and skeletal muscle (soleus)tissue compared to conventional treatment with alglucosidase alfa, andco-administration of miglustat with ATB200 showed a significant furtherreduction in lysosomal proliferation, approaching the levels seen inwild type (WT) mice. In addition, as seen in FIG. 21, administration ofATB200 showed a reduction in punctate glycogen levels in heart andskeletal muscle (soleus) tissue compared to conventional treatment withalglucosidase alfa, and co-administration of miglustat with ATB200showed a significant further reduction, again approaching the levelsseen in wild type (WT) mice.

As well, as seen in FIG. 22, co-administration of miglustat with ATB200significantly reduced the number of vacuoles in muscle fiber in thequadriceps of Gaa knockout mice compared to untreated mice and micetreated with alglucosidase alfa. As seen in FIG. 23, levels of both LC3II and p62 are increased in Gaa knockout mice compared to wild typemice, but are reduced significantly upon treatment with ATB200 andmiglustat, indicating that the increase in autophagy associated withacid α-glucosidase deficiency is reduced upon co-administration ofATB200 and miglustat. In addition, levels of the insulin-dependentglucose transporter GLUT4 and the insulin-independent glucosetransporter GLUT1 are increased in Gaa knockout mice compared to wildtype mice, but again, are reduced significantly upon treatment withATB200 and miglustat. The elevated GLUT4 and GLUT1 levels associatedwith acid α-glucosidase deficiency can contribute to increased glucoseuptake into muscle fibers and increased glycogen synthesis both basallyand after food intake. Thus, combination treatment with ATB200 andmiglustat has been found to improve skeletal muscle morphology andphysiology in a mouse model of Pompe disease.

Example 13 Toxicity of ATB200 Co-Administered with Miglustat inCynomolgus Monkeys

Naïve cynomolgus monkeys of Cambodian origin were assigned to dosegroups as indicated in Table 7. Animals were acclimated to the studyroom for 18 (females) to 19 (males) days. On the final day ofacclimation, animals weighed between 2.243 kg and 5.413 kg and were 2 to3 years of age.

TABLE 7 Dose Dose Number of Day 99 Level Conc. Animals Necropsy GroupTest Article Route (mg/kg) (mg/mL) (Male/Female) (Male/Female) 1 ControlIV 0 0 4/4 4/4 (Formulation Infusion Buffer) 2 Miglustat NG 25 2.5 4/44/4 ATB200 IV 50 5 Infusion 3 Miglustat NG 175 17.5 4/4 4/4 ATB200 IV100 10 Infusion 4 Miglustat NG 175 17.5 4/4 4/4 5 ATB200 IV 100 10 4/44/4 Infusion NG: nasogastric

Test dose levels were selected, based on previous studies in non-humanprimates, to provide exposures (AUC) comparable to or slightly above(for the 25 mg/kg miglustat and 50 mg/kg ATB200 group) or approximately10- and 3-fold higher than (for the 175 mg/kg miglustat and/or 100 mg/kgATB200 groups) the expected clinical AUCs in humans administered a doseof 260 mg miglustat and 20 mg/kg ATB200 as predicted from thepharmacokinetic models of Example 8 (approximately 20.9 hr·μg/mL andapproximately 1822 hr·μg/mL, respectively). In previous studies innon-human primates, an IV dose of 100 mg/kg ATB200 was found to resultin an AUC of 5330 hr·μg/mL, and an oral dose of 175 mg/kg of miglustatwas extrapolated to result in an AUC of 196 hr·μg/mL.

ATB200 is formulated in 25 mM sodium phosphate buffer, pH 6 containing2.92 mg/mL sodium chloride, 20 mg/mL mannitol, and 0.5 mg/mL polysorbate80 (formulation buffer). Test article (ATB200 or miglustat) and controlarticle/vehicle (formulation buffer) were administered once every otherweek for 13 weeks, starting on Day 1 and ending on Day 85. ATB200 andthe control article/vehicle were administered by 2 hour (±10 minute)intravenous (IV) infusion at 0 mg/kg (Group 1, control article), 50mg/kg (Group 2), or 100 mg/kg (Groups 3 and 5). Miglustat wasadministered nasogastrically in sterile water for injection, USP, at 25mg/kg (Group 2) or 175 mg/kg (Groups 3 and 4), 30 minutes (±2 minutes)prior to the start of the infusion for ATB200, when given incombination. The dosing volume across all groups was 10 mL/kg.

Parameters assessed during the in-life phase of the study included bodyweights, food consumption, clinical observations, detailed clinicalobservations, physical examinations, electrocardiography, ophthalmicassessments, clinical pathology (hematology, coagulation, serumchemistry), anti-drug antibody (ADA) assessment, neutralizing ADAassessment, urinalysis, and plasma toxicokinetics (TK) for miglustat andATB200 activity and total protein. Terminal necropsy of animals wasperformed on Day 99 (14 days after the last dose administration). Atnecropsy, gross observations and organ weights were recorded, andtissues were collected for microscopic examination.

All animals survived to the scheduled euthanasia and there were nochanges attributable to administration of ATB200, miglustat or to theco-administration of ATB200 and miglustat during the physicalexaminations or during assessment of food consumption, clinicalobservations, detailed clinical observations, body weights,ophthalmology, or ECG parameters. In addition, there was no ATB200,miglustat, or ATB200/miglustat-related changes in the urinalysis, serumchemistry, hematology, or coagulation parameters, or during assessmentof gross observations, organ weights, or histopathology.

Total Anti-Drug Antibody (ADA) and Neutralizing Antibody (NAb)

Total anti-drug antibody (ADA) and neutralizing antibody (NAb) levelsare measured in plasma. Blood samples (approximately 1.6 mL) werecollected in K₂EDTA tubes from all animals once during acclimation,predose (prior to administration of miglustat) and on Days 1, 85 and 99.Samples were maintained on wet ice until processed. Plasma was obtainedby centrifugation at 2° C. to 8° C. and aliquots (approximately 0.2 mL)were transferred to polypropylene vials, and stored frozen at −60° C. to−86° C. within one hour from blood collection. Analysis of samples forADA was conducted on samples collected from animals in Groups 1, 2, 3,and 5 (miglustat only samples were not analyzed). Analysis forneutralizing antibodies was conducted using an enzyme assay with thefluorogenic substrate 4-methylumbelliferyl-α-D-glucopyranoside(4MU-Glc).

All animals in the ATB200 dose Groups (Groups 2, 3, and 5) were positivefor anti-drug antibody (ADA) on Days 85 and 99 (100% incidence). Titersranged from 25600 to 409600 on Day 85 and from 51200 to 819200 on Day99. There was no obvious trend of titers increasing with increasingATB200 dose level. Five of 8 animals were positive for neutralizingantibody (NAb) in Group 2 (50 mg/kg ATB200 in combination with 25 mg/kgmiglustat) on Days 85 and 99. Two of 8 were positive for NAb on Day 85in Group 3 (100 mg/kg ATB200 in combination with 175 mg/kg miglustat)and 4 of 8 were positive on Day 99. Two of 8 were positive for NAb onDay 85 in Group 5 (100 mg/kg ATB200 monotherapy) and 3 of 8 werepositive on Day 99. There was no obvious effect of ADA on ATB200exposure or other TK parameters.

ATB200 Toxicokinetics

ATB200 toxicokinetics were measured in blood samples collected in K₂EDTAtubes from animals on Days 1 and 85 at the following time points:

For Groups 1, 2, 3, and 5: Predose (prior to administration ofmiglustat); 1 hour from initiation of infusion; 2 hours from initiationof infusion; 2.5 hours from initiation of infusion; 3 hours frominitiation of infusion; 4 hours from initiation of infusion; 6 hoursfrom initiation of infusion; 12 hours from initiation of infusion; 26hours from initiation of infusion; 168 hours from initiation ofinfusion; and 336 hours from initiation of infusion (collected prior todosing on Day 15); and

For Group 4: Predose (prior to administration of miglustat); 1.5 hourpost administration of miglustat; 2.5 hours post administration ofmiglustat; 3.5 hours post administration of miglustat; 4.5 hours postadministration of miglustat; 6.5 hours post administration of miglustat;12.5 hours post administration of miglustat; 26.5 hours postadministration of miglustat; 168.5 hours post administration ofmiglustat; and 336.5 hours post administration of miglustat (collectedprior to dosing on Day 15).

Plasma was obtained by centrifugation at 2° C. to 8° C. and aliquots(approximately 0.1 mL) were transferred to polypropylene vials, andstored frozen at −60° C. to −86° C. Analysis of ATB200 acidα-glucosidase activity and ATB200 total protein was conducted on the2-hour postdose samples from Group 1 animals and from all samplescollected from animals in Groups 2, 3, and 5. Total ATB200 protein wasmeasured by liquid chromatography coupled to tandem mass spectrometry(LC-MS/MS). Two signature peptides (TTPTFFPK and VTSEGAGLQLQK) were usedas a measure of ATB200. The results from these two peptides wereconsistent, indicating intact ATB200 was present in the analyzed plasmasamples. Acid α-glucosidase activity was assayed using the fluorogenicsubstrate 4-methylumbelliferyl-α-D-glucopyranoside (4MU-Glc).

Analysis of toxicokinetic (TK) data was performed on audited/verifieddata sets (concentration and time) from animals in Groups 2, 3, and 5using WinNonlin Phoenix, version 6.1 software (Pharsight Corporation).Noncompartmental analysis of individual subject plasma concentrationdata was used to estimate the TK parameters for acid α-glucosidaseactivity and ATB200 total protein (based on the two signature peptidesTTPTFFPK and VTSEGAGLQLQK) following IV infusion. The dose level wasentered as the actual ATB200 dose in mg, calculated based on eachindividual animal's dose volume, body weight, and the mean doseconcentration. The start time of each dosing (initiation of infusion forATB200) was set to zero for all profiles in the dosing regimen. Nominalsample collection times were used for all analyses. Thearea-under-the-plasma-concentration-time-curves (AUC_(0-t)) generatedfor ATB200 (total protein and activity assay datasets) were estimated bythe log-linear trapezoidal rule. The regression used to estimate λ_(z)was based on uniformly weighted concentration data.

The following parameters were calculated for each ATB200 data set(generated from the two signature peptides in the total ATB200 assay andfrom the ATB200 activity assay):

-   -   R²—the square of the correlation coefficient for linear        regression used to estimate λ_(zα). Used when a set number of        points are used to define the terminal phase (or specific time        range) of the concentration versus time profile;    -   R²adj—the square of the correlation coefficient for linear        regression used to estimate λ_(z), adjusted for the number of        points used in the estimation of λ_(zβ). Used when the number of        points used to define the terminal phase of the concentration        versus time profile may be variable;    -   No. points λ_(z)—number of points for linear regression analysis        used to estimate λ_(z);    -   λ_(zα)—elimination rate constant for the first three time points        after t_(max);    -   λ_(zβ)—terminal elimination rate constant;    -   t_(1/2α)—half-life based on the first three time points after        t_(max);    -   t_(1/2β)—terminal elimination half-life based on λ_(z)        (0.693/λ_(z));    -   t_(max)—time of maximal concentration of analyte in plasma;    -   C_(max)—maximal observed concentration of analyte in plasma;    -   AUC_(0-t)—Area-under-the-plasma-concentration-time-curve (AUC)        measured from time 0 (predose) through the time point with the        last measurable concentration;    -   AUC_(0-∞)—AUC extrapolated to time infinity;    -   AUC_(ext)—portion of AUC extrapolated to time infinity presented        as % of total AUC_(0-∞);    -   CL_(T)—total clearance (based on λ_(zβ)); based on total dose in        mg from actual body weight;    -   CL_(T)/F—total clearance (based on λ_(zβ)); based on total dose        in mg from actual body weight divided by the bioavailable        fraction;    -   V_(ss)—apparent volume of distribution at equilibrium;    -   V_(z)—volume of distribution based on the terminal phase (based        on λ_(zβ)); based on total dose in mg from actual body weight;    -   V_(z)/F—volume of distribution based on the terminal phase        (based on λ_(zβ)); based on total dose in mg from actual body        weight divided by the bioavailable fraction; and    -   Accumulation ratios—AR_(Cmax)=Ratio of C_(max) on Day 85 to Day        1; AR_(AUC)=Ratio of AUC_(0-t) on Day 85 to Day 1

ATB200 concentrations and TK parameters were similar between males andfemales. Plasma concentrations following a 50 mg/kg 2-hour IV ATB200infusion in combination with 25 mg/kg miglustat were measurable out tobetween 12 and 26 hours postdose. At the 100 mg/kg dose level (with orwithout 175 mg/kg miglustat), ATB200 concentrations were measurable outto 26 to 168 hours postdose. Toxicokinetic parameters for a single dose(Day 1) are shown in Table 8.

TABLE 8 Activity Total Protein Assay Assay Group Treatment ParameterUnits TTPTFFPK VTSEGAGLQLQK ATB200 2 50 mg/kg t_(max) hr 2.00 2.00 2.06ATB200 + C_(max) μg/mL 890 900 495 25 mg/kg AUC_(0-t) hr · μg/ 3060 30801700 miglustat mL t_(1/2α) hr 1.69 1.69 2.01 t_(1/2β) hr 1.70 1.71 1.92CL_(T) L/hr 0.058 0.058 0.106 V_(ss) L 0.145 0.144 0.266 V_(z) L 0.1440.143 0.296 3 100 mg/kg t_(max) hr 2.00 2.00 2.25 ATB200 + C_(max) μg/mL1960 1980 1150 175 mg/kg AUC_(0-t) hr · μg/ 10400 10400 6130 miglustatmL t_(1/2α) hr 2.77 2.77 3.07 t_(1/2β) hr 2.72 2.70 2.54 CL_(T) L/hr0.034 0.034 0.057 V_(ss) L 0.140 0.140 0.231 V_(z) L 0.133 0.133 0.210 5100 mg/kg t_(max) hr 1.88 1.88 1.94 ATB200 + C_(max) μg/mL 1690 16701270 Mono- AUC_(0-t) hr · μg/ 5490 5410 3230 therapy mL t_(1/2α) hr 1.561.55 1.28 t_(1/2β) hr 11.1 6.29 1.71 CL_(T) L/hr 0.105 0.105 0.140V_(ss) L 0.171 0.149 0.168 V_(z) L 0.729 0.401 0.383

Toxicokinetic parameters for repeat dosing (Day 85) are shown in Table9.

TABLE 9 Activity Total Protein Assay Assay Group Treatment ParameterUnits TTPTFFPK VTSEGAGLQLQK ATB200 2 50 mg/kg t_(max) hr 2.00 2.00 2.13ATB200 + C_(max) μg/mL 927 921 586 25 mg/kg AUC_(0-t) hr · μg/ 3700 37002390 miglustat mL t_(1/2α) hr 1.98 1.95 2.35 t_(1/2β) hr 2.38 2.40 2.31CL_(T) L/hr 0.049 0.049 0.076 V_(ss) L 0.147 0.147 0.223 V_(z) L 0.1680.168 0.254 3 100 mg/kg t_(max) hr 2.13 2.19 2.06 ATB200 + C_(max) μg/mL2270 2270 1600 175 mg/kg AUC_(0-t) hr · μg/ 13900 13800 9240 miglustatmL t_(1/2α) hr 3.62 3.72 3.34 t_(1/2β) hr 4.83 4.83 2.90 CL_(T) L/hr0.027 0.027 0.040 V_(ss) L 0.140 0.140 0.186 V_(z) L 0.174 0.174 0.165 5100 mg/kg t_(max) hr 2.13 2.13 2.00 ATB200 C_(max) μg/mL 2020 2010 1510Mono- AUC_(0-t) hr · μg/ 7830 7790 4890 therapy mL t_(1/2α) hr 1.93 1.881.44 t_(1/2β) hr 6.62 2.63 2.03 CL_(T) L/hr 0.045 0.045 0.070 V_(ss) L0.143 0.127 0.159 V_(z) L 0.396 0.170 0.205

The time to maximal ATB200 plasma concentration (t_(max)) wasapproximately 2 hours postdose in all three dose groups. The Day 1 andDay 85 ATB200 plasma concentrations and TK parameters, as measured bythe total ATB200 protein assay, were consistent between the twoevaluated signature peptides, TTPTFFPK and VTSEGAGLQLQK. Exposure, asmeasured by C_(max) and AUC_(0-t), was relatively lower when measured bythe acid α-glucosidase activity assay. This is to be expected, as thetotal protein assay measures concentration of both active and inactiveenzyme while the acid α-glucosidase activity assay measuresconcentration of active enzyme only. ATB200 exposure increased with dosebetween the 50 and 100 mg/kg dose levels. The mean Day 1 initialt_(1/2α) (males and females combined) based on the first three timepoints past t_(max) ranged from 1.28 to 3.07 hours. The mean Day 1terminal half-life (t_(1/2β)) ranged from 1.70 to 11.1 hours (the longert_(1/2β) values were influenced by the animals that had measurableconcentrations out to 168 hours postdose). A similar range of values wasobserved after the Day 85 dose. Little to no accumulation was observedwith repeated administration, once every other week. The addition of 175mg/kg miglustat to the 100 mg/kg ATB200 dose appeared to decrease ATB200clearance and increase plasma exposure approximately 2-fold, relative to100 mg/kg ATB200 monotherapy.

As no adverse test article-related changes were identified, theNo-Observed-Adverse-Effect-Level (NOAEL) for ATB200 in cynomolgusmonkeys when given once every other week for 13 weeks by 2 hourinfusion, with or without administration with miglustat, was 100mg/kg/infusion, the highest dosage tested. At this dose level, the meangender-averaged AUC_(0-t) and C_(max) (total protein) on Day 85 were7830 (TTPTFFPK) and 7790 (VTSEGAGLQLQK) hr·μg/mL and 2020 (TTPTFFPK) or2010 (VTSEGAGLQLQK) μg/mL, respectively, for ATB200 alone and 13900(TTPTFFPK) or 13800 (VTSEGAGLQLQK) hr·μg/mL and 2270 (both peptides)μg/mL, respectively, in combination with 175 mg/kg Miglustat.

Miglustat Toxicokinetics

Miglustat toxicokinetics were measured in blood samples collected inK₂EDTA tubes from animals on Days 1 and 85 at the following time points:

For Groups 1, 2, 3, and 5: Predose (prior to administration ofmiglustat); 15 minutes after administration of miglustat; 0 hour (priorto initiation of infusion); 0.5 hours from initiation of infusion; 1hour from initiation of infusion; 2 hours from initiation of infusion; 4hours from initiation of infusion; 6 hours from initiation of infusion;12 hours from initiation of infusion; 26 hours from initiation ofinfusion; 50 hours from initiation of infusion; and 74 hours frominitiation of infusion; and

For Group 4: Predose (prior to administration of miglustat); 15 minutespost administration of miglustat; 30 minutes post administration ofmiglustat; 1 hour post administration of miglustat; 1.5 hours postadministration of miglustat; 2.5 hours post administration of miglustat;4.5 hours post administration of miglustat; 6.5 hours postadministration of miglustat; 12.5 hours post administration ofmiglustat; 26.5 hours post administration of miglustat; 50.5 hours postadministration of miglustat; and 74.5 hours post administration ofmiglustat.

Plasma was obtained by centrifugation at 2° C. to 8° C. and aliquots(approximately 0.2 mL) were transferred to polypropylene vials, andstored frozen at −60° C. to −86° C. Analysis of miglustat concentrationwas carried out using a LC-MS/MS method analogous to that described foranalysis of duvoglustat concentration by Richie Khanna, Allan C. PoweJr., Yi Lun, Rebecca Soska, Jessie Feng, Rohini Dhulipala, MichelleFrascella, Anadina Garcia, Lee J. Pellegrino, Su Xu, Nastry Brignol,Matthew J. Toth, Hung V. Do, David J. Lockhart, Brandon A. Wustman,Kenneth J. Valenzano. “The Pharmacological Chaperone AT2220 Increasesthe Specific Activity and Lysosomal Delivery of Mutant AcidAlpha-Glucosidase, and Promotes Glycogen Reduction in Transgenic MouseModel of Pompe Disease.” PLOS ONE (1 Jul. 2014) 9(7): e102092.

Analysis of toxicokinetic (TK) data for miglustat was performed onaudited/verified data sets (concentration and time) from animals inGroup 2, 3, and 4 using WinNonlin Phoenix®, version 6.1 software(Pharsight Corporation). Noncompartmental analysis of individual plasmaconcentration data was used to estimate the TK parameters. Miglustat TKparameters were estimated by the log-linear trapezoidal rule. Theregression used to estimate λ_(z) was based on uniformly weightedconcentration data. The following parameters were calculated:

-   -   R²adj—the square of the correlation coefficient for linear        regression used to estimate λ_(z), adjusted for the number of        points used in the estimation of λ_(z). Used when the number of        points used to define the terminal phase of the concentration        versus time profile may be variable;    -   No. points λ_(z)—number of points for linear regression analysis        used to estimate λ_(z);    -   λ_(z)—the terminal elimination rate constant;    -   t_(1/2)—terminal elimination half-life based on λ_(z)        (0.693/λ_(z));    -   t_(max)—time of maximal concentration of analyte in plasma;    -   C_(max) maximal observed concentration of analyte in plasma;    -   AUC_(0-t)—Area-under-the-plasma-concentration-time-curve (AUC)        measured from time 0 (predose) through the time point with the        last measurable concentration;    -   AUC_(0-∞)—AUC extrapolated to time infinity;    -   AUC_(ext)—portion of AUC extrapolated to time infinity presented        as % of total AUC_(0-∞);    -   CL_(T)/F—total clearance divided by the bioavailable fraction        based on total dose in mg from actual body weight;    -   V_(z)/F—volume of distribution based on the terminal phase        divided by the bioavailable fraction based on total dose in mg        from actual body weight;    -   Accumulation ratios—AR_(Cmax)=Ratio of _(Cmax) on Day 85 to Day        1; and AR_(AUC)=Ratio of AUC_(0-t) on Day 85 to Day 1.

There was no consistent effect of sex on miglustat TK parameters.Miglustat plasma concentrations following either a 25 mg/kg nasogastric(NG) administration in combination with 50 mg/kg ATB200, or a 175 mg/kgNG administration (with or without 100 mg/kg ATB200), were measurable to74.5 hours (the last measured time point). Toxicokinetic parameters fora single dose (Day 1) and for repeat dosing (Day 85) are shown in Table10.

TABLE 10 Miglustat Assay Group Treatment Parameter Units Day 1 Day 85 250 mg/kg t_(max) hr 2.06 2.88 ATB200 + C_(max) ng/mL 7430 7510 25 mg/kgAUC_(0-t) hr · ng/mL 47300 49100 miglustat t_(1/2) hr 7.44 8.23 CL_(T)/FL/hr 1.92 1.99 V_(z)/F L 20.5 23.3 3 100 mg/kg t_(max) hr 2.69 3.56ATB200 + C_(max) ng/mL 20400 22000 175 mg/kg AUC_(0-t) hr · ng/mL 182000216000 miglustat t_(1/2) hr 6.85 7.86 CL_(T)/F L/hr 3.22 3.62 V_(z)/F L32.3 39.1 4 175 mg/kg t_(max) hr 3.00 4.13 Miglustat C_(max) ng/mL 1640014700 Monotherapy AUC_(0-t) hr · ng/mL 173000 204000 t_(1/2) hr 6.866.66 CL_(T)/F L/hr 3.67 3.49 V_(z)/F L 35.9 33.8

The t_(max) ranged from approximately 2 to 4 hours postdose. Miglustatexposure increased with dose between the 25 and 175 mg/kg dose levels.The mean t_(1/2) (males and females combined) was consistent on Days 1and 85 and ranged from 6.66 to 8.23 hours. Little to no accumulation wasobserved with repeat once every other week NG administration. There wasno observable effect of ATB200 co-administration on overall miglustatexposure (i.e., AUC_(0-t)) or TK parameters.

As no adverse test article-related changes were identified, theNo-Observed-Adverse-Effect-Level (NOAEL) for miglustat in cynomolgusmonkeys when given once every other week for 13 weeks nasogastrically,with or without administration with ATB200, was 175 mg/kg/dose, thehighest dosage tested. At this dose level, the mean gender-averagedAUC_(0-t) and C_(max) on Day 85 were 204000 hr·ng/mL and 14700 ng/mL,respectively, for miglustat alone and 216000 hr·ng/mL and 22000 ng/mL,respectively, in combination with 100 mg/kg ATB200.

Example 14 Protocol for Clinical Study of Recombinant Acid α-Glucosidase(ATB200) Administered Alone and Co-Administered with Miglustat

Study Design:

This is an open-label, fixed-sequence, ascending-dose, first-in-humanstudy to evaluate the safety, tolerability, and pharmacokinetics (PK) ofintravenous (IV) recombinant acid α-glucosidase (ATB200, lyophilizedpowder reconstituted with sterile water for injection and diluted with0.9% sodium chloride for injection) alone and when co-administered withoral miglustat (hard gelatin capsules, 65 mg). The study will beconducted in 2 stages. In Stage 1, safety, tolerability, and PK will beevaluated following sequential single ascending doses of ATB200,administered every 2 weeks as an approximately 4 hour intravenousinfusion, for 3 dosing periods at 5, 10, and 20 mg/kg. In Stage 2,safety, tolerability, and PK will be evaluated following single- andmultiple-ascending dose combinations: 20 mg/kg ATB200 co-administeredevery 2 weeks with 130 mg miglustat (two 65 mg capsules), taken orally 1hour prior to an approximately 4 hour intravenous infusion of ATB200,for 3 doses followed by 20 mg/kg ATB200 co-administered with 260 mgmiglustat (four 65 mg capsules), taken orally 1 hour prior to anapproximately 4 hour intravenous infusion of ATB200, for 3 doses.

Twelve enzyme replacement therapy (ERT)-experienced subjects with Pompedisease (approximately 6 ambulatory and 6 nonambulatory) will enrollinto Stage 1. Those same subjects will continue the study in Stage 2. Atleast 4 ambulatory subjects will be enrolled and dosed beforenonambulatory subjects are enrolled. ERT-experienced (ambulatory)subjects are defined as those who have been on ERT for 2 to 6 yearsprior to enrollment, who are able to walk at least 200 meters in thesix-minute walk test (6MWT), and have an FVC of 30-80% of predictednormal value. ERT-experienced (nonambulatory) subjects are defined asthose who are completely wheelchair bound, unable to walk unassisted,and have been on ERT for years prior to enrollment. Treatment assignmentis shown in Table 11.

TABLE 11 Stage 2 Stage 1 Period 4 Period 5 Number Period 1 Period 2Period 3 Multiple Dose Multiple Dose of Population: ERT Single SingleSingle Co- Co- Subjects Experienced Dose Dose Dose administrationadministration 12 ~6 ambulatory, 5 mg/kg 10 mg/kg 20 mg/kg 20 mg/kg 20mg/kg ~6 nonambulatory ATB200 ATB200 ATB200 ATB200 + ATB200 + 130 mg 260mg miglustat miglustat ERT = enzyme replacement therapy.

Subjects will be required to fast at least 2 hours before and 2 hoursafter administration of oral miglustat. IV infusion of ATB200 shouldstart 1 hour after oral administration of miglustat.

Study Procedures

The study consists of Screening, Baseline, Stage 1 (3-period,fixed-sequence, single-ascending-dose of ATB200 alone), and Stage 2(2-period, fixed-sequence, multiple-dose of 20 mg/kg ATB200co-administered with multiple-ascending-doses of miglustat).

Screening:

All subjects will provide informed consent and undergo review ofeligibility criteria. Assessments for all subjects include medicalhistory including prior infusion-associated reactions (IARs) and historyof falls; review of prior and concomitant medications and nondrugtherapies; vital signs (heart rate [HR], respiration rate [RR], bloodpressure [BP], and temperature); height; weight; comprehensive physicalexamination (PE); 12-lead electrocardiogram (ECG); clinical safetylaboratory assessments (serum chemistry, hematology, and urinalysis);urine pregnancy test; urine sample for hexose tetrasaccharide (Hex4);and GAA genotyping (for subjects unable to provide GAA genotyping reportat screening). A blood sample will also be obtained for exploratoryassessment of immunogenicity (total and neutralizing antibodies,exploratory cytokines/other biomarkers of immune system activation,cross reactivity to alglucosidase alfa, and immunoglobulin E [IgE]) ifneeded. A subject who meets all of the inclusion criteria and none ofthe exclusion criteria will be assigned to Stage 1 as described in Table11.

Baseline:

Safety assessments for all subjects include review of eligibilitycriteria; medical history including infusion associated reactions (IARs)and history of falls, adverse event (AE) and serious AE (SAE) inquiry,review of prior and concomitant medications and nondrug therapies; vitalsigns (HR, RR, BP, and temperature); weight; brief PE; ECG; Rasch-builtPompe-specific activity (R-PAct) scale; Rotterdam Handicap Scale; andFatigue Severity Scale; clinical safety laboratory assessments (serumchemistry, hematology, and urinalysis); urine pregnancy test;pharmacodynamic (PD) assessments (Hex4 and creatinine phosphokinase[CPK]); immunogenicity assessments (total and neutralizing antibodies,antibody cross-reactivity with alglucosidase alfa, exploratory cytokinesand other biomarkers of immune system activation, cross reactivity toalglucosidase alfa, and IgE if needed); pulmonary function tests (PFTs);motor function tests; and muscle strength tests for all subjects.

Stage 1, Periods 1, 2, and 3:

This stage will include:

-   -   Safety: review of AEs, including serious adverse events (SAEs)        and IARs; review of concomitant medications and nondrug        therapies; vital signs (HR, RR, BP, and temperature); brief PE;        ECG; clinical safety laboratory assessments (serum chemistry,        hematology, and urinalysis); and urine pregnancy test    -   PD: urinary Hex4 and serum CPK    -   Immunological: blood samples for anti-recombinant acid        α-glucosidase antibody titers (anti-recombinant acid        α-glucosidase total and neutralizing antibody titers and        antibody cross-reactivity with alglucosidase alfa) and blood        samples for measurement of pro-inflammatory cytokines and other        biomarkers of immune system activation. If needed, IgE        measurements will also be performed.    -   Serial 24-hour pharmacokinetics (PK): During Period 1 (Visit 3,        Day 1), Period 2 (Visit 4, Day 15), and Period 3 (Visit 5, Day        29), blood sampling for plasma acid α-glucosidase activity        levels and total acid α-glucosidase protein concentrations will        be taken for all subjects.

Stage 2, Periods 4 and 5:

-   -   Safety: review of AEs, including SAEs and IARs; review of        concomitant medications and nondrug therapies; vital signs (HR,        RR, BP, and temperature); weight; PE; ECG; clinical safety        laboratory assessments (serum chemistry, hematology, and        urinalysis); and urine pregnancy test    -   PD: urinary Hex4 and serum CPK    -   Immunological: blood samples for anti-recombinant acid        α-glucosidase antibody titers (anti-recombinant acid        α-glucosidase total and neutralizing antibody titers, and        antibody cross-reactivity with alglucosidase alfa) and blood        samples for measurement of pro-inflammatory cytokines and other        biomarkers of immune system activation. If needed, IgE        measurements will also be performed.    -   Serial 24-hour PK: During Period 4 (Visit 6, Day 43 and Visit 8,        Day 71) and Period 5 (Visit 9, Day 85 and Visit 11, Day 113),        blood sampling for plasma acid α-glucosidase activity levels,        total acid α-glucosidase protein concentrations, and miglustat        concentrations will be taken for all subjects.

End of Pharmacokinetic Phase:

-   -   Safety: review of AEs, including SAEs and IARs; review of        concomitant medications and nondrug therapies; vital signs (HR,        RR, BP, and temperature); weight; PE; ECG; clinical safety        laboratory assessments (serum chemistry, hematology, and        urinalysis); and urine pregnancy test    -   PD: urinary Hex4 and serum CPK    -   Immunological: blood samples for anti-recombinant acid        α-glucosidase antibody titers (anti-recombinant acid        α-glucosidase total and neutralizing antibody titers, and        antibody cross-reactivity with alglucosidase alfa) and blood        samples for measurement of pro-inflammatory cytokines and other        biomarkers of immune system activation. If needed, IgE        measurements will also be performed.

Subjects who prematurely withdraw from the study will come in for anEarly Termination visit and will undergo all of the assessments that areto be performed at the End of PK visit. No study drug will beadministered. If any of the sentinel subjects withdraw prematurely fromthe study, that subject will be replaced by the next ambulatory subjectenrolled in the study (e.g., if Subject 1 withdraws, Subject 3[ambulatory] will replace that subject as a sentinel subject).

Subjects who complete this study and/or other subjects who qualify willbe offered the opportunity to participate in a long-term extension studyand will continue to be assessed for safety and tolerability of ATB200co-administered with miglustat. In addition, functional assessmentsrelevant to Pompe disease will be performed in the extension study atregular intervals.

Safety Monitoring

Safety will be monitored by the Medical Monitor and the investigators onan ongoing basis, and on a regular basis by a Safety Steering Committee(SSC).

Sentinel Dosing

The first 2 ambulatory subjects in this study will be the sentinelsubjects for the study and will be the first 2 subjects dosed in eachperiod of the study (Periods 1 to 5). In the event that a sentinelsubject prematurely withdraws from the study, he/she will be replaced byanother ambulatory subject. Note: At least 4 ambulatory subjects will bedosed with 5 mg/kg ATB200 before any nonambulatory subjects can bedosed.

In Stage 1 (Periods 1, 2, and 3), subjects will be dosed with singleascending doses of ATB200 (5 mg/kg [Period 1], 10 mg/kg [Period 2], and20 mg/kg [Period 3]).

Following the dosing of the 2 sentinel subjects for each study period inStage 1, an evaluation of the available safety data (PE, vital signs,AEs, infusion reactions, ECG, and available locally performed laboratorytests) will be performed within 24 to 48 hours by the Medical Monitorand the investigators. The SSC will convene for a formal safety reviewwhen central safety laboratory data are available for both sentinelsubjects at each dose level. If there SSC determines that there are nosafety concerns that preclude dosing at the dose assigned for thatperiod, 10 additional subjects will be enrolled and dosed. The SSC willalso convene for a safety review when safety data (including centrallaboratory safety data) for all subjects at all 3 Stage-1 dose levelsare available.

In Stage 2 (Periods 4 and 5), the 2 sentinel subjects will be dosed, andsafety will be assessed after the first dose as for each period inStage 1. If the SSC determines that there are no safety concerns thatpreclude additional dosing at 20 mg/kg ATB200 co-administered with 130mg miglustat (Period 4) or 20 mg/kg ATB200 co-administered with 260 mgmiglustat (Period 5), 10 additional subjects will receive 3 biweeklydoses at the dose assigned for that period. The SSC will reconvene whenall safety data (including central safety laboratory data) are availablefor all subjects at the end of Stage 2. The SSC will also convene ad hocin case of an SAE or an identified safety concern. The SSC may recommendany of the following reviews:

-   -   Continue the study without modifications    -   Continue the study with modifications (amendment)    -   Temporarily halt dosing    -   Permanently stop dosing

If in the opinion of the SSC there are no AEs or safety concerns in thesentinel subjects that might preclude continued study dosing, dosingwill continue for all remaining subjects at that dose level. Subjectsafety will continue to be closely monitored by the Medical Monitor andstudy investigators on an ongoing basis, and at regular intervals by theSSC.

Number of Subjects (Planned):

Twelve adult ERT-experienced subjects with Pompe disease (approximately6 ambulatory and 6 nonambulatory) will enroll into Stage 1. Those samesubjects will continue the study in Stage 2.

Diagnosis and Eligibility Criteria:

At the Screening Visit, adult ERT-experienced subjects with Pompedisease will be evaluated using the eligibility criteria outlined below.Each subject must meet all of the inclusion criteria and none of theexclusion criteria. Waivers of inclusion/exclusion criteria are notpermitted.

Inclusion Criteria

ERT-Experienced Subjects (Ambulatory)

-   -   1. Male and female subjects between 18 and 65 years of age,        inclusive;    -   2. Subject must provide signed informed consent prior to any        study-related procedures;    -   3. Subjects of childbearing potential must agree to use        medically accepted methods of contraception during the study and        for 30 days after last co-administration of ATB200+miglustat;    -   4. Subject has a diagnosis of Pompe disease based on documented        deficiency of acid α-glucosidase enzyme activity or by GAA        genotyping;    -   5. Subject has received ERT with alglucosidase alfa for the        previous 2-6 years;    -   6. Subject is currently receiving alglucosidase alfa at a        frequency of once every other week;    -   7. Subject has received and completed the last two infusions        without a drug-related adverse event resulting in dose        interruption;    -   8. Subject must be able to walk 200-500 meters on the 6MWT; and    -   9. Upright forced vital capacity (FVC) must be 30% to 80% of        predicted normal value.

ERT-Experienced Subjects (Nonambulatory)

-   -   10. Male and female subjects between 18 and 65 years of age,        inclusive;    -   11. Subject must provide signed informed consent prior to any        study-related procedures;    -   12. Subjects of childbearing potential must agree to use        medically accepted methods of contraception during the study and        for 30 days after last co-administration of ATB200+miglustat;    -   13. Subject has a diagnosis of Pompe disease based on documented        deficiency of acid α-glucosidase enzyme activity or by GAA        genotyping;    -   14. Subject has received ERT with alglucosidase alfa for years;    -   15. Subject is currently receiving alglucosidase alfa at a        frequency of once every other week;    -   16. Subject has received and completed the last two infusions        without a drug-related adverse event resulting in dose        interruption; and    -   17. Subject must be completely wheelchair-bound and unable to        walk unassisted.

Exclusion Criteria

ERT-Experienced Subjects (Ambulatory)

-   -   1. Subject has received any investigational therapy for Pompe        disease, other than alglucosidase alfa within 30 days prior to        the Baseline Visit, or anticipates doing so during the study;    -   2. Subject has received treatment with prohibited medications        (miglitol (eg, Glyset®); miglustat (eg, Zavesca®); acarbose (eg,        Precose®, Glucobay®); voglibose (eg, Volix®, Vocarb®, and        Volibo®); albuterol and clenbuterol; or any        investigational/experimental drug) within 30 days of the        Baseline Visit;    -   3. Subject, if female, is pregnant or breastfeeding at        screening;    -   4. Subject, whether male or female, is planning to conceive a        child during the study;    -   5. Subject requires invasive ventilatory support;    -   6. Subject uses noninvasive ventilatory support hours a day        while awake;    -   7. Subject has a medical or any other extenuating condition or        circumstance that may, in the opinion of the investigator, pose        an undue safety risk to the subject or compromise his/her        ability to comply with protocol requirements;    -   8. Subject has a history of anaphylaxis to alglucosidase alfa;    -   9. Subject has a history of high sustained anti-recombinant acid        α-glucosidase antibody titers;    -   10. Subject has a history of allergy or sensitivity to miglustat        or other iminosugars;    -   11. Subject has a known history of autoimmune disease including        lupus, autoimmune thyroiditis, scleroderma, or rheumatoid        arthritis; and    -   12. Subject has a known history of bronchial asthma.

ERT-Experienced Subjects (Nonambulatory)

-   -   13. Subject has received any investigational therapy for Pompe        disease, other than alglucosidase alfa within 30 days prior to        the Baseline Visit, or anticipates to do so during the study;    -   14. Subject has received treatment with prohibited medications        (miglitol (eg, Glyset®); miglustat (eg, Zavesca®); acarbose (eg,        Precose®, Glucobay®); voglibose (eg, Volix®, Vocarb®, and        Volibo®); albuterol and clenbuterol; or any        investigational/experimental drug) within 30 days of the        Baseline Visit;    -   15. Subject, if female, is pregnant or breastfeeding at        screening;    -   16. Subject, whether male or female, is planning to conceive a        child during the study;    -   17. Subject has a medical or any other extenuating condition or        circumstance that may, in the opinion of the investigator, pose        an undue safety risk to the subject or compromise his/her        ability to comply with protocol requirements;    -   18. Subject has a history of anaphylaxis to alglucosidase alfa;    -   19. Subject has a history of high sustained anti-recombinant        acid α-glucosidase antibody titers;    -   20. Subject has a history of allergy or sensitivity to miglustat        or other iminosugars;    -   21. Subject has a known history of autoimmune disease including        lupus, autoimmune thyroiditis, scleroderma, or rheumatoid        arthritis; and    -   22. Subject has a known history of bronchial asthma.

Investigational Product, Dosage, and Mode of Administration:

Stage 1 (Consists of 3 Dosing Periods 2 Weeks Apart)

-   -   Period 1: a single-dose IV infusion of 5 mg/kg ATB200;    -   Period 2: a single-dose IV infusion of 10 mg/kg ATB200 to all        subjects who have completed Period 1; and    -   Period 3: a single-dose IV infusion of 20 mg/kg ATB200 to all        subjects who have completed Period 2.

Stage 2 (Consists of 2 Dosing Periods, Each Comprising 3 Study DrugDoses, 2 Weeks Apart)

-   -   Period 4: 130 mg of miglustat will be administered orally 1 hour        before a single dose IV infusion of 20 mg/kg ATB200 to all        subjects who have completed Period 3 (repeated every 2 weeks for        a total of 3 administrations); and    -   Period 5: 260 mg of miglustat will be administered orally 1 hour        before a single dose IV infusion of 20 mg/kg ATB200 to all        ERT-experienced subjects who have completed Period 4 (repeated        every 2 weeks for a total of 3 administrations).

Note: Subjects are required to fast at least 2 hours before and 2 hoursafter administration of oral miglustat.

Total Duration of Study: Up to 22 weeks (up to 4 weeks screening periodfollowed by approximately 18 weeks of study treatment [Stages 1 and 2])

Duration of single-dose PK observation (Stage 1, Periods 1, 2, and 3): 6weeks

Duration of multiple-dose PK observation (Stage 2, Periods 4 and 5): 12weeks

Duration of safety, tolerability, and immunogenicity observation(Periods 1, 2, 3, 4, and 5): 18 weeks

Criteria for Evaluation:

Primary:

Safety Assessments:

-   -   PEs    -   Vital signs, including body temperature, RR, HR, and BP    -   AEs, including IARs    -   12-Lead ECG    -   Clinical safety laboratory assessments: serum chemistry,        hematology, and urinalysis

PK of Plasma ATB200 and Miglustat:

-   -   Plasma acid α-glucosidase activity levels and total acid        α-glucosidase protein concentrations PK parameters: maximum        observed plasma concentration (C_(max)), time to reach the        maximum observed plasma concentration (t_(max)), area under the        plasma-drug concentration time curve from Time 0 to the time of        last measurable concentration (AUC_(0-t)), area under the        plasma-drug concentration time curve from Time 0 extrapolated to        infinity (AUC_(0-∞)), half-life (t_(1/2)), and total clearance        following IV administration (CL_(T))    -   Ratios of plasma acid α-glucosidase activity and total acid        α-glucosidase protein C_(max) and AUC_(0-∞) for all dose        regimens    -   Plasma miglustat PK parameters: C_(max), t_(max), AUC_(0-t),        AUC_(0-∞), and t_(1/2), apparent total clearance of drug        following oral administration (CL_(T)/F), and terminal phase        volume of distribution following oral administration (Vz/F) for        each dose level    -   Ratios of plasma miglustat C_(max) and AUC_(0-∞) for each dose        level

Functional Assessments (Performed at Baseline)

For Ambulatory Subjects

-   -   Motor Function Tests        -   Six minute Walk Test (6MWT)        -   10-Meter Walk Test        -   Gait, Stairs, Gower, and Chair score        -   Timed Up and Go (TUG)    -   Muscle Strength Test (medical research criteria [MRC] and        hand-held dynamometer) for both upper and lower limbs    -   PFTs (FVC, MIP, MEP, and SNIP)

For Nonambulatory Subjects

-   -   Muscle Strength Test—Upper Limbs Only        -   MRC and hand-held dynamometer performed for upper limbs only    -   Pulmonary function tests (PFTs) (forced vital capacity [FVC],        maximum inspiratory pressure [MIP], maximum expiratory pressure        [MEP], and sniff nasal inspiratory pressure [SNIP])

Patient-Reported Outcomes (Performed at Baseline)

-   -   Fatigue Severity Scale    -   Rotterdam Handicap Scale    -   Rasch-built Pompe-specific activity (R-PAct)

Exploratory

-   -   Anti-ATB200 antibody titers (total and neutralizing)    -   Cross-reactivity of anti-recombinant acid α-glucosidase        antibodies to alglucosidase alfa    -   Pro-inflammatory cytokines and other biomarkers of immune system        activation    -   PD markers (Hex4 and CPK)

Methods of Analysis:

Statistical Methods:

Descriptive statistics on PK parameters will be provided. Summarystatistics will be provided for all variables that are not PKparameters. Dose proportionality assessment on acid α-glucosidaseactivity and total acid α-glucosidase protein exposure (C_(max),AUC_(0-t), and AUC_(0-∞)) ratios of 5, 10, and 20 mg/kg ATB200 alone.Analysis of variance (ANOVA) on acid α-glucosidase activity and totalacid α-glucosidase protein exposure (C_(max), AUC_(0-t), and AUC_(0-∞))ratios of 20 mg/kg ATB200 alone versus 20 mg/kg ATB200+130 mg miglustat,and versus 20 mg/kg ATB200+260 mg miglustat within each population andoverall. ANOVA on acid α-glucosidase activity and total acidα-glucosidase protein exposure (C_(max), AUC_(0-t), and AUC_(0-∞))ratios between ambulatory and nonambulatory subjects for 20 mg/kgATB200+130 mg miglustat and 20 mg/kg ATB200+260 mg miglustat. Doseproportionality assessment for exposure ratios (C_(max), AUC_(0-t), andAUC_(0-∞)) between 130 mg and 260 mg miglustat within each subjectpopulation and overall. The effect of immunogenicity results on PK, PD,and safety will be evaluated.

Interim Analyses:

An interim analysis will be performed when at least 50% (n=6) of thesubjects have completed Stage 2 of the study. Up to 2 additional interimanalyses may be performed in the study.

Initial PK Results:

The PK summary of GAA activity and GAA total protein for subjects isshown in Tables 12 and 13, respectively.

In Tables 12-15 and FIGS. 24-26, the single dose (SD) measurements weretaken after a single administration of miglustat and ATB200, and themultiple dose (MD) measurements were taken after the third biweeklyadministration of miglustat and ATB200.

TABLE 12 Dose mg/kg ATB200 + mg at_(1/2) ^(a) βt_(1/2) ^(a) t_(max) ^(b)C_(max) ^(c) AUC_(0-t) ^(c) AUC_(0-∞) ^(c) AUC_(0-∞)/D^(c) CL_(T) ^(a)V_(ss) ^(a) miglustat (hr) (hr) (hr) (ug/mL) (hr * ug/mL) (hr * ug/mL)(hr * ug/mL/mg) (L/hr) (L)  5 1.06 3.15 3.5 (3.5-4.0) 53.7 193 193 0.444(15.4) 2.27 5.61 (9.7) (5.3) (20.4) (22.5) (22.5) (15.9) (21.2) 10 1.262.73 3.75 (3.5-4.5)  115 447 448 0.523 (17.5) 1.93 5.39 (22.2) (18.2)(28.3) (30.7) (30.6) (15.0) (21.2) 20 1.36 2.16 4.0 (3.5-4.0) 256 10201021 0.596 (30.1) 1.76 5.01 (25.7) (10.2) (30.4) (37.4) (37.4) (37.5)(28.0) 20 + 130 1.84 2.49 4.5 (4.0-5.0) 234 1209 1211 0.707 (23.7) 1.455.32 Single (16.0) (9.9) (36.0) (29.9) (29.9) (25.8) (24.8) Dose 20 +130 1.90 2.53 4.0 (3.5-5.0) 230 1180 1183 0.690 (15.1) 1.46 5.55Multiple (7.5) (11.9) (20.2) (19.1) (19.0) (14.4) (14.2) Dose 20 + 2602.39 2.70 4.0 (4.0-4.5) 228 1251 1256 0.733 (15.8) 1.38 5.71 Single(11.5) (10.8) (26.0) (17.4) (17.2) (17.3) (20.2) Dose ^(a)Arithmeticmean (CV %) ^(b)Median (min-max) ^(c)Geometric mean (CV %)

TABLE 13 Dose mg/kg ATB200 + mg αt_(1/2) ^(a) βt_(1/2) ^(a) t_(max) ^(b)C_(max) ^(c) AUC_(0-t) ^(c) AUC_(0-∞) ^(c) AUC_(0-∞)/D^(c) CL_(T) ^(a)V_(ss) ^(a) miglustat (hr) (hr) (hr) (ug/mL) (hr * ug/mL) (hr * ug/mL)(hr * ug/mL/mg) (L/hr) (L)  5 1.02 1.83 4.0 (3.5-4.0) 61.1 215 218 0.511(7.3)  1.97 4.57 (3.0) (13.8) (20.0) (17.1) (17.0) (7.7) (6.8) 10 1.361.99 4.0 143 589 594 0.694 (12.3) 1.45 3.90 (5.3) (56.9) (19.5) (16.6)(16.6) (13.4) (14.5) 20 1.65 2.62 4.0 338 1547 1549 0.904 (12.8) 1.113.49 (12.3) (18.5) (11.1) (12.1) (12.1) (14.4) (11.6) 20 + 130 1.79 2.634.0 322 1676 1680 0.980 (15.0) 1.03 3.78 Single (10.7) (6.6) (18.2)(14.9) (14.8) (17.6) (12.2) Dose 20 + 130 1.99 2.47 4.0 (3.5-5.0) 3551800 1804  1.05 (12.9) 0.96 3.70 Multiple (10.2) (4.2) (16.5) (12.7)(12.7) (13.7) (10.8) Dose 20 + 260 2.35 2.73 4.0 350 1945 1953  1.14(15.8) 0.89 3.63 Single (13.9) (10.4) (14.2) (15.1) (15.0) (15.7) (16.3)Dose ^(a)Arithmetic mean (CV %) ^(b)Median (min-max) ^(c)Geometric mean(CV %)

FIG. 24A shows the concentration-time profiles of mean plasma GAAactivity after doses of 5 mg/kg, 10 mg/kg and 20 mg/kg ATB200. FIG. 24Balso provides the concentration time-profiles profiles of mean plasmaGAA activity after doses of 5 mg/kg, 10 mg/kg and 20 mg/kg ATB200, butthe plasma GAA activity is displayed on a logarithmic scale. As can beseen from FIGS. 24A-24B and Table 12, ATB200 demonstrated slightlygreater than dose proportional exposures for plasma GAA activity.

FIG. 24C shows the concentration-time profiles of mean plasma GAAactivity after doses of 20 mg/kg ATB200 alone, as well as 20 mg/kg ofATB200 and 130 or 260 mg of miglustat. FIG. 24D also provides the meanplasma GAA activity after doses of 20 mg/kg ATB200 alone, with 130 mgmiglustat or 260 mg miglustat, but the plasma GAA activity is displayedon a logarithmic scale.

FIG. 25A shows the concentration-time profiles of mean plasma GAA totalprotein after doses of 5 mg/kg, 10 mg/kg and 20 mg/kg ATB200. FIG. 25Balso provides the concentration time-profiles profiles of mean plasmaGAA total protein after doses of 5 mg/kg, 10 mg/kg and 20 mg/kg ATB200,but the plasma GAA total protein is displayed on a logarithmic scale. Ascan be seen from FIGS. 25A-25B and Table 13, ATB200 demonstratedslightly greater than dose proportional exposures for plasma GAA totalprotein.

FIG. 25C shows the concentration-time profiles of mean plasma GAA totalprotein after doses of 20 mg/kg ATB200 alone, 20 mg/kg of ATB200 and 130mg of miglustat, and 20 mg/kg of ATB200 and 260 mg of miglustat. FIG.25D also provides the mean plasma GAA total protein after doses of 20mg/kg ATB200 alone, with 130 mg miglustat or 260 mg miglustat, but theplasma GAA total protein is displayed on a logarithmic scale.

As shown in Table 13, co-administration of miglustat increased total GAAprotein plasma half-life by approximately 30% relative to ATB200administered alone. Volume of distribution ranged from 3.5 to 5.7 L forall treatments, suggesting that the glycosylation of ATB200 enablesefficient distribution of ATB200 to tissues.

The PK summary for miglustat is shown in Table 14.

TABLE 14 Dose βt_(1/2) ^(a) t_(max) ^(b) C_(max) ^(c) C_(max)/BW^(c)AUC_(0-t) ^(c) AUC_(0-∞) ^(c) AUC_(0-∞)/BW^(c) V_(z)/F^(a) CL/F^(a) mg(hr) (hr) (ug/mL) (ng/mL/kg) (hr * ug/mL) (hr * ug/mL) (hr * ng/mL/kg)(L) (L/hr) 130 4.5 2.75 1647 19.2 (23.9) 12620 13157 154 (29.7) 65.49.93 Single (37.0) (1.5-3.5) (22.1) (13.1) (13.1) (41.9) (13.7) Dose 1305.6 3.0  1393 16.3 (36.4) 11477 12181 142 (26.9) 88.1 10.8 Multiple(12.5) (1.5-3.5) (36.8) (18.0) (16.4) (26.1) (16.2) Dose 260 5.5 2.753552 41.5 (33.8) 26631 28050 325 (30.8) 79.2 9.51 Single (25.9)(1.0-5.0) (30.2) (25.1) (22.9) (55.3) (27.6) Dose ^(a)Arithmetic mean(CV %) ^(b)Median (min-max) ^(c)Geometric mean (CV %)

FIG. 26 shows the concentration-time profile of miglustat in plasma inhuman subjects after dosing of 130 mg or 260 mg of miglustat.

As can be seen from Table 14 and FIG. 26, plasma miglustat, administeredorally 1 hour prior to ATB200 infusion, reached peak concentrations 2hours into the infusion and demonstrated dose-proportional kinetics.

An analysis was performed on various portions of the plasmaconcentration curves for GAA activity and total protein to determinepartial AUCs. Table 15 provides a summary of partial AUCs from0-t_(max), t_(max)-6 h, t_(max)-10 h, t_(max)-12 h and t_(max)-24 h forGAA activity and total protein.

TABLE 15 Arithmetic Mean pAUC (ng * hr/mL) at Time Post-Dose (N = 4)t_(max)- Analyte Treatment 0-t_(max) t_(max)-6 h t_(max)-10 h t_(max)-12h 24 h GAA 20 mg/kg 428 382 606 630 654 Activity GAA 20 mg/kg + 456 415722 770 832 Activity 130 mg Single Dose GAA 20 mg/kg + 423 392 689 737796 Activity 130 mg Multiple Dose GAA 20 mg/kg + 423 536 924 996 1094Activity 260 mg Single Dose Total 20 mg/kg 621 603 943 981 1040 ProteinTotal 20 mg/kg + 565 614 1041 1106 1189 Protein 130 mg Single Dose Total20 mg/kg + 630 612 1079 1154 1244 Protein 130 mg Multiple Dose Total 20mg/kg + 679 824 1411 1518 1665 Protein 260 mg Single Dose

As can be seen from Table 15, GAA activity percent mean increases ofpAUCt_(max-24 h) for 20 mg/kg plus miglustat relative to 20 mg/kg ATB200alone were 21.4%, 17.8%, 40.2%, for 130 mg SD, 130 mg MD, and 260 mg SD,respectively.

Similarly, GAA total protein percent mean increases of pAUCt_(max-24 h)for 20 mg/kg plus miglustat relative to 20 mg/kg ATB200 alone were12.5%, 16.4%, 37.5%, for 130 mg SD, 130 mg MD, and 260 mg SD,respectively.

Thus, the partial AUC analysis demonstrates that co-administration ofmiglustat significantly increases the terminal phase partial AUC(t_(max-24 h)) of ATB200 by approximately 15% for doses of 130 mg ofmiglustat and approximately 40% for 260 mg of miglustat.

Initial Biomarker Results

Alanine aminotransferase (ALT), aspartate aminotransferase (AST) andcreatine phosphokinase (CPK) levels were monitored in human patientsthat switched from Lumizyme® to ATB200. The patients received ascendingdoses of ATB200 (5, 10 and 20 mg/kg) followed by co-administration ofATB200 (20 mg/kg) and miglustat (130 and 260 mg). High levels of CPKenzyme may indicate injury or stress to muscle tissue, heart, or brain.Elevated ALT and AST are markers of liver and muscle damage from Pompedisease, respectively. The initial analysis of the ALT, AST and CPKlevels are shown in FIGS. 38-41.

As can be seen from FIGS. 38-41, two patients showed early trend towardimprovement in all three biomarkers and two patients remained stable.One patient had 44%, 28% and 34% reductions in CPK, AST and ALTrespectively. Another patient had 31%, 22% and 11% reductions in CPK,AST and ALT respectively.

Thus far, there have been no serious adverse events (SAEs). AEs weregenerally mild and transient. There have been no infusion-associatedreactions to date following 100+ infusions in all patients enrolled. Allpatients had anti-rhGAA antibodies at baseline which remained generallystable. Cytokines remained low and stable during infusions.

Example 15 GAA and LAMP1 Levels in Wild-Type and Pompe Fibroblasts

Immunofluorescence microscopy was utilized for detecting GAA and LAMP1levels in wild-type fibroblasts and Pompe fibroblasts with a commonsplicing mutation. As shown in FIG. 27, GAA is in distinct lysosomalcompartments in wild-type fibroblasts. FIG. 27 also shows an abundantGAA signal in the Pompe fibroblasts, and that both the GAA and LAMP1signals in Pompe fibroblasts appear to be localized to the ER and Golgi,rather than distal lysosomes. This is evidence of altered GAA proteintrafficking in Pompe fibroblasts.

Example 16 Improvement of Cellular Dysfunction and Muscle Function inGaa-Knockout Mice

Impairment of lysosomal glycogen catabolism due to GAA deficiency hasbeen shown to cause substantial cellular dysfunction as evidenced bypronounced, persistent autophagy and proliferation and accumulation ofmembrane-bound intracellular compartments filled with accumulatedglycogen (N. Raben et al.). Our immunohistologic data indicate thatprotein trafficking is significantly altered for many proteins includingseveral key proteins that are vital for muscle membrane stability suchas dystrophin, α- and β-dystroglycan, various sarcoglycans and othersthat comprise the dystrophin glycoprotein complex as well as proteinsinvolved in muscle repair such as dysferlin. These key muscle proteinsrequire proper protein trafficking to the muscle cell membrane wherethey function. As shown in FIG. 28, our immunohistologic data revealthat an appreciable fraction of these key muscle proteins have anintracellular localization in muscles of Gaa knockout (KO) mouse modelof Pompe disease. These data suggest that mistrafficking of these keymuscle proteins may induce a pseudo-muscular dystrophy that ultimatelylead to muscle weakness and disrepair. Alglucosidase alfa (Myozyme®) andATB200 with and without 10 mg/kg miglustat were evaluated in Gaa KO miceat equivalent ERT dose (20 mg/kg) via an every other week dosingschedule. After 2 administrations, alglucosidase alfa modestly reducedaccumulated lysosomal glycogen in skeletal muscles (FIGS. 32A-32C) andhad negligible effects towards reducing autophagy (FIGS. 30A-30B) orlysosomal proliferation (FIGS. 29A-29B) as compared to vehicle-treatedmice. In contrast, substantially better lysosomal glycogen clearance wasobserved with ATB200/miglustat under identical conditions (FIGS.32A-32D). ATB200/miglustat also appeared to improve overall musclephysiology as evidenced by reduced LC3 II levels (FIGS. 30A-30B), awell-established autophagy biomarker and by clearance of accumulatedintracellular vesicles stained with LAMP1 (FIGS. 29A-29B), a knownresident lysosomal integral membrane protein and dysferlin (FIGS.31A-31B), a known cell surface protein involved in muscle repair.Further, ATB200/miglustat significantly improved the muscle architecturethat resembled muscle fibers of wild-type mice. Also, FIGS. 29-32 showthat two different batches of ATB200 (earlier and later generationmanufacturing processes) produced comparable results. In FIGS.32A-32D, * indicates statistically significant compared to Myozyme®alone.

Example 17 Muscle Function in Gaa-Knockout Mice

In longer-term studies of 12 biweekly administrations, 20 mg/kg ATB200plus 10 mg/kg miglustat progressively increased functional musclestrength in Gaa KO mice from baseline as measured by both grip strengthand wire hang tests (FIGS. 33A-33B). Alglucosidase alfa(Lumizyme®)-treated mice receiving the same ERT dose (20 mg/kg) wereobserved to decline under identical conditions throughout most of thestudy (FIGS. 33A-33B). As with the shorter-term study, ATB200/miglustathad substantially better glycogen clearance after 3 months (FIGS.34A-34C) and 6 months (FIGS. 34D-G) of treatment than alglucosidasealfa. ATB200/miglustat also reduced autophagy and intracellularaccumulation of LAMP1 and dysferlin after 3 months of treatment (FIG.35) compared to alglucosidase alfa. In FIG. 33A, * indicatesstatistically significant compared to Lumizyme® alone (p<0.05, 2-sidedt-test). In FIGS. 34A-34G, * indicates statistically significantcompared to Lumizyme® alone (p<0.05, multiple comparison using Dunnett'smethod under one-way ANOVA analysis).

Taken together, these data indicate that ATB200/miglustat wasefficiently targeted to muscles to reverse cellular dysfunction andimprove muscle function. Importantly, the apparent improvements inmuscle architecture and reduced autophagy and intracellular accumulationof LAMP1 and dysferlin may be good surrogates for improved musclephysiology that correlate with improvements in functional musclestrength. These results suggest that monitoring autophagy and these keymuscle proteins may be a rational, practical method to assess theeffectiveness therapeutic treatments for Pompe disease in Gaa KO micethat may prove to be useful biomarkers from muscle biopsies in clinicalstudies.

FIG. 40 shows that 6 months of ATB200 administration with or withoutmiglustat lowered intracellular accumulation of dystrophin in Gaa KOmice. There was a greater reduction for dystrophin accumulation forATB200±miglustat than with Lumizyme®.

Example 18 Effect of Sialic Acid Content on ATB200 in Gaa-Knockout Mice

Two batches of ATB200 with different sialic acid content were evaluatedfor pharmacokinetics and efficacy in in Gaa KO mice. Table 16 provides asummary of the characteristics for the two batches.

TABLE 16 Characteristic Batch A Batch B Sialic Acid 4.0 mol/mol protein5.4 mol/mol protein M6P content 3.3 mol/mol protein 2.9 mol/mol proteinSpecific activity 115831 (nmol 120929 (nmol 4 mu/mg protein/hr) 4 mu/mgprotein/hr) CIMPR binding K_(d) = 2.7 nM K_(d) = 2.9 nM

As can be seen from Table 16, Batch B had a higher sialic acid contentthan Batch A, but a slightly lower M6P content than Batch A.

FIG. 36 shows the shows the concentration-time profiles of GAA activityin plasma in Gaa KO mice after a single IV bolus dosing of the ATB200.The half-life of Batches A and B are provided in Table 17 below.

TABLE 17 Half-life (hr) Mean ± SEM Batch A 0.50 ± 0.02 Batch B 0.60 ±0.03

As can be seen from Table 17, Batch B had a lower half-life than BatchA. Although the decrease in half-life was modest, this decrease inhalf-life was statistically significant (p<0.05 in 2-sided t-test).

In a related study, IV bolus tail vein injections of ATB200 (Batches Aand B) and Lumizyme® were given to Gaa KO mice every other week for atotal of 2 injections. Glycogen levels in tissues were measured 14 daysafter last administration. As shown in FIGS. 37A-37D, Batch B wasgenerally more effective in reducing glycogen than Batch A at similardoses. Both Batch A and Batch B were superior to Lumizyme® in reducingglycogen. In FIGS. 37A-37D, * indicates statistically significantcompared to Lumizyme® (p<0.05, t-test) and ̂ indicates statisticallysignificant comparison of Batch A and Batch B at same dose (p<0.05,t-test).

The embodiments described herein are intended to be illustrative of thepresent compositions and methods and are not intended to limit the scopeof the present invention. Various modifications and changes consistentwith the description as a whole and which are readily apparent to theperson of skill in the art are intended to be included. The appendedclaims should not be limited by the specific embodiments set forth inthe examples, but should be given the broadest interpretation consistentwith the description as a whole.

Patents, patent applications, publications, product descriptions,GenBank Accession Numbers, and protocols are cited throughout thisapplication, the disclosures of which are incorporated herein byreference in their entireties for all purposes.

1. A method of treating Pompe disease in a patient in need thereof, themethod comprising administering miglustat to the patient in combinationwith a recombinant acid α-glucosidase, wherein the recombinant acidα-glucosidase is expressed in Chinese hamster ovary (CHO) cells andcomprises an increased content of N-glycan units bearing one or twomannose-6-phosphate residues when compared to a content of N-glycanunits bearing one or two mannose-6-phosphate residues of alglucosidasealfa.
 2. The method according to claim 1 wherein the recombinant humanacid α-glucosidase comprises a sequence at least 95% identical to SEQ IDNO: 1 or SEQ ID NO:
 5. 3. The method according to claim 1 wherein atleast 30% of molecules of the recombinant human acid α-glucosidasecomprise one or more N-glycan units bearing one or twomannose-6-phosphate residues.
 4. The method according to claim 1 whereinthe recombinant human acid α-glucosidase comprises on average from 0.5to 7.0 moles of N-glycan units bearing one or two mannose-6-phosphateresidues per mole of recombinant human acid α-glucosidase.
 5. The methodaccording to claim 1 wherein the recombinant human acid α-glucosidasecomprises on average at least 2.5 moles of mannose-6-phosphate residuesper mole of recombinant human acid α-glucosidase and at least 4 moles ofsialic acid residues per mole of recombinant human acid α-glucosidase.6. The method according to claim 1 wherein the recombinant human acidα-glucosidase comprises seven potential N-glycosylation sites, at least50% of molecules of the recombinant human acid α-glucosidase comprise anN-glycan unit bearing two mannose-6-phosphate residues at the firstsite, at least 30% of molecules of the recombinant human acidα-glucosidase comprise an N-glycan unit bearing one mannose-6-phosphateresidue at the second site, at least 30% of molecules of the recombinanthuman acid α-glucosidase comprise an N-glycan unit bearing twomannose-6-phosphate residue at the fourth site, and at least 20% ofmolecules of the recombinant human acid α-glucosidase comprise anN-glycan unit bearing one mannose-6-phosphate residue at the fourthsite.
 7. The method according to claim 1 wherein the recombinant humanacid α-glucosidase is produced by Chinese hamster ovary cell lineGA-ATB-200 or ATB-200-001-X5-14 or a subculture thereof.
 8. The methodaccording to claim 1 wherein the recombinant human acid α-glucosidase isadministered intravenously at a dose of about 5 mg/kg to about 20 mg/kgand the miglustat is administered orally at a dose of about 200 mg toabout 600 mg.
 9. The method according to claim 8 wherein the miglustatis administered prior to administration of the recombinant human acidα-glucosidase.
 10. The method according to claim 9 wherein the miglustatis administered about one hour prior to administration of therecombinant human acid α-glucosidase.
 11. The method according to claim1 wherein the recombinant human acid α-glucosidase is administeredintravenously at a dose of about 5 mg/kg to about 20 mg/kg and themiglustat is administered orally at a dose of about 233 mg to about 500mg.
 12. The method according to claim 1 wherein the recombinant humanacid α-glucosidase is administered intravenously at a dose of about 5mg/kg to about 20 mg/kg and the miglustat is administered orally at adose of about 50 mg to about 200 mg.
 13. The method according to claim 1wherein the recombinant human acid α-glucosidase is administeredintravenously at a dose of about 20 mg/kg and the miglustat isadministered orally at a dose of about 260 mg.
 14. The method accordingto claim 13 wherein the miglustat is administered prior toadministration of the recombinant human acid α-glucosidase.
 15. Themethod according to claim 14 wherein the miglustat is administered aboutone hour prior to administration of the recombinant human acidα-glucosidase.
 16. A kit for combination therapy of Pompe disease in apatient in need thereof, the kit including a pharmaceutically acceptabledosage form comprising miglustat, a pharmaceutically acceptable dosageform comprising a recombinant human acid α-glucosidase, and instructionsfor administering the pharmaceutically acceptable dosage form comprisingmiglustat and the pharmaceutically acceptable dosage form comprising therecombinant acid α-glucosidase to a patient in need thereof; wherein therecombinant acid α-glucosidase is expressed in Chinese hamster ovary(CHO) cells and comprises an increased content of N-glycan units bearingone or two mannose-6-phosphate residues when compared to a content ofN-glycan units bearing one or two mannose-6-phosphate residues ofalglucosidase alfa.
 17. The kit according to claim 16 wherein thepharmaceutically acceptable dosage form comprising the recombinant humanacid α-glucosidase is configured for intravenous administration at adose of about 5 mg/kg to about 20 mg/kg and pharmaceutically acceptabledosage form comprising miglustat comprises a dose of about 200 mg toabout 600 mg and is configured for oral administration.
 18. The kitaccording to claim 17 wherein the instructions comprise instructions toadminister the miglustat about one hour prior to administration of therecombinant human acid α-glucosidase.
 19. The kit according to claim 16wherein the pharmaceutically acceptable dosage form comprising therecombinant human acid α-glucosidase is configured for intravenousadministration at a dose of about 5 mg/kg to about 20 mg/kg and thepharmaceutically acceptable dosage form comprising miglustat comprises adose of about 50 mg to about 200 mg and is configured for oraladministration.
 20. The kit according to claim 16 wherein thepharmaceutically acceptable dosage form comprising the recombinant humanacid α-glucosidase is configured for intravenous administration at adose of about 20 mg/kg and the pharmaceutically acceptable dosage formcomprising miglustat comprises a dose of about 260 mg and is configuredfor oral administration.
 21. The kit according to claim 20 wherein theinstructions comprise instructions to administer the pharmaceuticallyacceptable dosage form comprising miglustat about one hour prior toadministration of the pharmaceutically acceptable dosage form comprisingthe recombinant human acid α-glucosidase.